Detecting pax2 for the diagnosis of breast cancer

ABSTRACT

A method for monitoring breast conditions in a subject is disclosed. The method comprises determining a Paired Box 2 gene-to-beta defensin-1 gene (PAX2-to-DEFB1) expression ratio (the “Donald Predictive Factor” or “DPF”) in cells obtained from the breast of the subject, wherein the PAX2-to-DEFB1 expression ratio is correlated with breast conditions. Also disclosed is a kit for monitoring breast conditions and determining drug resistance.

This application is a continuation of U.S. patent application Ser. No.12/546,292, filed Aug. 24, 2009, which is a continuation-in-part of theU.S. patent application Ser. No. 12/440,193, filed Mar. 13, 2009, nowU.S. Pat. No. 8,088,603, as a national stage application ofPCT/US2008/051168, filed Jan. 16, 2008, which claims priority of U.S.Provisional Application No. 60/885,142, filed Jan. 16, 2007. Theentirety of all of the aforementioned application is incorporated hereinby reference.

FIELD

The present application generally relates to medical diagnosis and, inparticular, to methods for diagnosing cancerous conditions in varioustissues.

BACKGROUND

Breast cancer is the most common cause of cancer in women and the secondmost common cause of cancer death in women in the U.S. While themajority of new breast cancers are diagnosed as a result of anabnormality seen on a mammogram, a lump or change in consistency of thebreast tissue can also be a warning sign of the disease. Heightenedawareness of breast cancer risk in the past decades has led to anincrease in the number of women undergoing mammography for screening,leading to detection of cancers in earlier stages and a resultantimprovement in survival rates. Still, breast cancer is the most commoncause of death in women between the ages of 45 and 55.

Breast cancer may be classified into several stages. Stage 0 iscarcinoma (including lobular carcinoma and ductal carcinoma) in situ.Stage I is an early stage of invasive breast cancer. The tumor is nomore than 2 centimeters across. Cancer cells have not spread beyond thebreast. Stage II tumors include tumors that are no more than 2centimeters across but has spread to the lymph nodes under the arm,tumors that are between 2 and 5 centimeters and may have spread to thelymph nodes under the arm, and tumors that are larger than 5 centimeters(2 inches) but has not spread to the lymph nodes under the arm. StageIII is locally advanced cancer. It is further divided into Stage IIIA,IIIB, and IIIC. Stage IV is distant metastatic cancer. The cancer hasspread to other parts of the body. Early-stage treatment options aredifferent from late-stage options.

It is known that many types of cancer are caused by genetic aberrations,i.e., mutations. The accumulation of mutations and the loss of cellularcontrol functions cause progressive phenotypic changes from normalhistology to early pre-cancer such as intraepithelial neoplasia (IEN) toincreasingly severe IEN to superficial cancer and finally to invasivedisease. Although this process can be relatively aggressive in somecases, it generally occurs relatively slowly over years and evendecades. Oncogene addiction is the physiologic dependence of cancercells on the continued activation or over expression of single oncogenesfor maintaining the malignant phenotype. This dependence occurs in themilieu of the other changes that mark neoplastic progression.

The long period of progression to invasive cancer provides anopportunity for clinical intervention. Therefore, it is important toidentify biomarkers that are indicative of pre-cancerous conditions sothat treatment measures can be taken to prevent or delay the developmentof invasive cancer.

SUMMARY

One aspect of the present invention relates to a method for monitoringbreast conditions in a subject. The method comprises determining aPaired Box 2 gene-to-beta defensin-1 gene (PAX2-to-DEFB1) expressionratio in cells obtained from the breast of the subject, wherein thePAX2-to-DEFB1 expression ratio is correlated with breast conditions andmay serve as a prognosticator used for determining course of treatment.

In one embodiment, a PAX2-to-DEFB1 expression ratio of 100:1 or higheris indicative of the presence of breast cancer in the subject, and aPAX2-to-DEFB1 expression ratio less than 100:1 is indicative of thepresence of non-cancerous or pre-cancerous breast condition in thesubject.

In another embodiment, the determining step comprises determining theexpression level of PAX2 gene relative to the expression level of acontrol gene, determining the expression level of DEFB1 gene relative tothe expression level of the same control gene; and determining thePAX2-to-DEFB1 expression ratio based on the expression levels of PAX2and DEFB1.

In one embodiment, the method further comprising determining anoestrogen receptor/progesterone receptor/human epidermal growth factorreceptor 2 (ER/PR/HER2) status in cells obtained from the breast tissuewith the breast condition.

Another aspect of the present invention relates to a kit for monitoringbreast conditions. In one embodiment, the kit for monitoring breastconditions comprises: one or more pairs of oligonucleotide primers fordetecting PAX2 expression in a tissue sample, one or more pairs ofoligonucleotide primers for detecting DEFB1 expression in the tissuesample, and instructions on how to determine the PAX2-to-DEFB1expression ratio in a tissue sample using the primers. In anotherembodiment, the one or more pairs of oligonucleotide primers fordetecting PAX2 expression comprising an oligonucleotide primer pairselected from the group consisting of SEQ ID NOS: 43 and 47, SEQ ID NOS:44 and 48, and SEQ ID NOS: 45 and 49.

In another embodiment, the one or more pairs of oligonucleotide primersfor detecting DEFB1 expression comprising SEQ ID NOS: 35 and 37.

In another embodiment, the kit further comprises one or more pairs ofcontrol oligonucleotide primers. In one embodiment, the one or morepairs of control oligonucleotide primers comprise oligonucleotideprimers for detecting expression of β-actin expression. In a preferredembodiment, the oligonucleotide primers for detecting expression ofβ-actin expression comprise SEQ ID NOS: 34 and 36.

In another embodiment, the one or more pairs of control oligonucleotideprimers comprise oligonucleotide primers for detecting expression ofGAPDH expression. In a preferred embodiment, the oligonucleotide primersfor detecting expression of GAPDH expression comprise SEQ ID NOS: 42 and46.

In another related embodiment, the kit further comprises one or morereagents for PCR reaction.

In yet another related embodiment, the kit further comprises one or morereagents for RNA extraction.

In another embodiment, the kit for monitoring breast conditionscomprises an oligonucleotide microarray having oligonucleotide probesfor detecting PAX2 and DEFB1 expression and instructions on how todetermine the PAX2-to-DEFB1 expression ratio in a tissue sample usingthe oligonucleotide microarray.

In a related embodiment, the kit further comprises reagents forextracting RNA from a tissue sample.

Another aspect of the invention relates to a method for determining atreatment regimen for a subject with a breast condition. The methodincludes the step of determining the expression level of PAX2 generelative to the expression level of a control gene in cells obtainedfrom a breast tissue with the breast condition in said subject, anddetermining a treatment regimen for said subject based on the relativeexpression level of the PAX2 gene.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of thedisclosed method and compositions and together with the description,serve to explain the principles of the disclosed method andcompositions.

FIG. 1 shows quantitative RT-PCR (QRT-PCR) analysis of beta-defensin-1(DEFB1) expression. In order to verify induction of DEFB1 expression,QRT-PCR was performed. FIG. 1A shows DEFB1 relative expression levelscompared in clinical samples from 6 patients that underwent radicalprostatectomies. FIG. 1B shows DEFB1 relative expression levels comparedin benign and malignant prostatic clinical samples, hPrEC cells and inprostate cancer cell lines before and after DEFB1 induction. FIG. 1Cshows DEFB1 relative expression levels analyzed in benign tissue,malignant tissue and prostate intraepithelial neoplasia (PIN) in asingle tissue section. FIG. 1D shows DEFB1 expression in benign tissue,malignant tissue and PIN in one patient compared to the average DEFB1expression level found in benign tissue.

FIG. 2 shows microscopic analysis of DEFB1 induced changes in membraneintegrity and cell morphology. Cell morphology of DU145, PC3 and LNCaPwas analyzed by phase contrast microscopy after 48 hours of DEFB1induction. Membrane ruffling is indicated by black arrows and apoptoticbodies are indicated white arrows.

FIG. 3 shows analysis of DEFB1 Cytotoxicity in Prostate Cancer Cells.The prostate cell lines DU145, PC3 and LNCaP were treated with PonA toinduce DEFB1 expression for 1-3 days after which MTT assay was performedto determine cell viability. Results represent mean±s.d., n=9.

FIG. 4 shows induction of cell death in DU145 and PC3 cells by DEFB1.DEFB1 expression was induced in prostate cancer cell lines DU145 (A) andPC3 (B) and then subjected to annexin V/FITC/propidium iodide stainingand flow cytometric analysis. Cells positive for propidium iodide andannexin V were considered apoptotic. Times of induction are shown undereach panel. Numbers next to the boxes for each time point represent thepercentages of propidium iodide (PI)− annexin V+ cells (lower rightquadrant), and PI+ annexin V+ cells (upper right quadrant). The data arefrom a single experiment that is representative of three separateexperiments.

FIG. 5 shows pan-caspase analysis following DEFB1 induction. DU145 andPC3 cells were stained with FAM-VAD-FMK-labeled fluoromethyl ketone todetect caspase activity. Cells were visible under DIC for eachcondition. Confocal microscopic analysis revealed no caspase staining incontrol DU145 (B), PC3 cells (F) and LNCaP (J). Cells treated with PonAfor 24 hours to induce DEFB1 revealed caspase activity in DU145 (D) andPC3 (H). No caspase activity was detected in LNCaP (L).

FIG. 6 shows silencing of paired box homeotic gene 2 (PAX2) proteinexpression following PAX2 siRNA Treatment. FIG. 6A shows Western blotanalysis of PC3 and DU145 cells transfected with PAX2 siRNA duplex atday zero (lane 1), day two (lane 2), and day four (lane 3). FIG. 6Bshows Western blot analysis of PC3 and DU145 cells transfected with PAX2siRNA duplex at day zero (lane 1), day two (lane 2), day four (lane 3)and day 6 (lane 4). PAX2 protein was undetectable as early as after fourdays of treatment (lane 3) in DU145 cells and after six days oftreatment in PC3. Blots were stripped and re-probed for β-actin as aninternal control.

FIG. 7 shows analysis of prostate cancer cells growth after treatmentwith PAX2 siRNA. Phase contrast microscopic analysis of DU145, PC3 andLNCaP at 6 days in the presence of normal growth media. Treatment withnegative control siRNA had no effect on the cells. However, there was asignificant reduction in cell number in all three lines followingtreatment with PAX2 siRNA.

FIG. 8 shows analysis of cell death following siRNA silencing of PAX2.Prostate cancer cell lines PC3, DU145, and LNCaP were treated with 0.5μg of a pool of four PAX2 siRNA's or four non-specific control siRNA'sfor 2, 4 or 6 days after which MTT assay was done to determine cellviability. Results represent mean±s.d., n=9.

FIG. 9 shows analysis of caspase activity. DU145, PC3 and LNCaP cellswere stained with carboxyfluorescein-labeled fluoromethyl ketone todetected caspase activity following treatment with PAX2 siRNA. Confocalmicroscopic analysis of untreated and treated cells show cells werevisible with DIC. Analysis under fluorescence revealed no caspasestaining in control DU145 (B), PC3 cells (F) and LNCaP cells (J).However, cell treated with PAX2 siRNA induced caspase activity in DU145(D), PC3 (H) and LNCaP (L).

FIG. 10 shows analysis of apoptotic factors following PAX2 siRNAtreatment. Changes in expression of pro-apoptotic factors were comparedin untreated control cells and in cells treated for six days with PAX2siRNA. FIG. 10A shows Bcl-2-associated X protein (BAX) expression levelsincreased in DU145, PC3 and LNCaP. FIG. 10B shows BH3 interacting domaindeath agonist (BID) expression increased in DU145 and LNCaP, but changein PC3. FIG. 10C shows Bcl-2-associated death promoter (BAD) expressionlevels increased in all three cell lines.

FIG. 11 shows model of PAX2 binding to DNA recognition sequence. ThePAX2 transcriptional repressor binds to a CCTTG (SEQ ID NO: 1)recognition site immediately adjacent to the DEFB1 TATA box preventingtranscription and DEFB1 protein expression. Inhibition of PAX2 proteinexpression allows normal DEFB1 expression.

FIG. 12 illustrates the DEFB1 reporter construct. The DEFB1 promoterconsisting of the first 160 bases upstream of the mRNA start site wasPCR amplified from DU145 cell and ligated into the pGL3 luciferasereporter plasmid.

FIG. 13 shows inhibition of PAX2 results in DEFB1 Expression. DU145,PC3, LNCaP and HPrEC were treated for 48 hours with PAX2 siRNA. QRT-PCRanalysis before treatment showed no DEFB1 expression in DU145, PC3 andLNCaP. However, DEFB1 expression was restored following treatment in alllines. There was no change in DEFB1 expression following siRNA treatmentof PAX2-null HprEC.

FIG. 14 shows inhibition of PAX2 results in increased DEFB1 promoteractivity. PC3 promoter/pGL3 and DU145 promoter/pGL3 construct weregenerated and were transfected into PC3 and DU145 cells, respectively.Promoter activity was compared before and after PAX2 inhibition by siRNAtreatment. DEFB1 promoter activity increased 2.65-fold in DU145 and 3.78fold in PC3 following treatment.

FIG. 15 shows ChIP analysis of PAX2 binding to DEFB1 promoter. ChIPanalysis was performed on DU145 and PC3 cells. Followingimmunoprecipitation with an anti-PAX2 antibody, PCR was performed todetect the DEFB1 promoter region containing the GTTCC (SEQ ID NO: 2)PAX2 recognition site. This demonstrates that the PAX2 transcriptionalrepressor is bound to the DEFB1 promoter in prostate cancer cell lines.

FIG. 16 shows predicted structure of the PrdPD and PrdHD with DNA. Thecoordinates of the structures of the PrdPD bound to DNA (Xu et al.,1995) and the PrdHD bound to DNA (Wilson et al., 1995) were used toconstruct a model of the two domains as they bound to a PHO site. Theindividual binding sites are abutted next to each other with a specificorientation as indicated. The RED domain is oriented based on the PrdPDcrystal structure.

FIG. 17 shows comparison of consensus sequences of different paireddomains. At the top of the Figure is drawn a schematic representation ofprotein±DNA contacts described in the crystallographic analysis of thePrd-paired-domain±DNA complex. Empty boxes indicate a-helices, shadedboxes indicates b-sheets and a thick line indicate a b-turn. Contactingamino acids are shown by single-letter code. Only direct amino acid±basecontacts are shown. Empty circles indicate major groove contacts whilered arrows indicate minor groove contacts. This scheme is aligned to allknown consensus sequences for paired-domain proteins (top strands onlyare shown). Vertical lines between consensus sequences indicateconserved base-pairs. Numbering of the positions is shown at the bottomof the Figure.

FIG. 18 shows targeting PAX2 as a chemopreventive strategy. AberrantPAX2 expression is an early event in the initiation and progression ofcancer. Inhibition of PAX2 during dysplasia or other precancerous stagecan be used for cancer prevention.

FIG. 19 shows effect of angiotensin II (Ang II) on PAX2 expression inDU145 Cells. In order to determine the effect of AngII on PAX2expression, DEFB1 protein levels was monitored following treatment. HerePAX2 expression levels increased as early as 4 hours and persisted until48 hours.

FIG. 20A shows effect of Losartan (Los) on PAX2 expression in DU145.DU145 cells were treated with the angiotensin II type 1 receptor (ATR1)blocker Losartan. QRT-PCR revealed that PAX2 message levels weredecreased by at least half following treatment. FIG. 20B shows effect ofan angiotensin II type 2 receptor (ATR2) blocker on PAX2 Expression inDU145. To determine the effect of the ATR2 receptor on PAX2 expression,DU145 cells were treated with the ATR2 receptor blocker PD123319. Here,PAX2 expression was increased 7 to 8-fold.

FIG. 21 shows Los blocks AngII effect on PAX2 expression in DU145.Treatment of DU145 cells with 5 μM of AngII for 72 hours resulted in a2-fold increase in PAX2 expression. In addition, treatment with 10 μMfor 72 hours resulted in more than a 3-fold increase in expression.Treatment of cells with 5 μM of Losartan suppressed proliferation by50%. In addition, treatment with Losartan for 30 min prior to treatmentwith AngII blocked the effect of AngII on proliferation.

FIG. 22 shows AngII increases DU145 cell proliferation. Treatment ofDU145 cells with 5 μM of AngII for 72 hours resulted in a 2-foldincrease in proliferation. In addition, treatment with 10 μM for 72hours resulted in more than a 3-fold increase in proliferation.

FIG. 23 shows effect of Los and MAP Kinase inhibitors on PAX2 expressionin DU145 cells. FIG. 23A shows treatment of DU145 cells with Losartansuppresses phosphor-ERK 1/2 and PAX2 expression; FIG. 23B shows MEKkinase inhibitors and AICAR suppresses PAX2 protein expression; FIG. 23Cshows MEK kinase inhibitors and Losartan suppresses phospho-STAT3protein expression.

FIG. 24 shows effect of Los and MEK kinase inhibitors on PAX2 activationin DU145 cells. FIG. 24A shows treatment of DU145 cells with inhibitorsof AT1R signaling resulted in a decrease in phosphor-PAX2 protein levelswhich is the active form of PAX2. In addition, treatment with the AMPkinase inducer AICAR resulted in suppressed PAX2 expression. FIG. 24Bshows inhibition of AT1R signaling with Los decreased phopho-JNK levels.However, AngII increased phosphor-JNK protein levels.

FIG. 25 shows AngII increases PAX2 and decreases DEFB1 expression inhPrEC cells. To determine the effect of AngII on PAX2 levels in hPrEC,cells were treated for 72 and 96 hours and PAX2 and DEFB1 expression wasexamined by QRT-PCR. Here, AngII treatment resulted in dramaticincreases in PAX2 to levels similar to PC3 prostate cancer cells.Conversely, DEFB1 expression was reduced significantly after AngIItreatment.

FIG. 26 shows schematic of AngII signaling and PAX2 prostate cancer.PAX2 expression in prostate cancer cells is regulated by the AT1Rsignaling pathway. Specifically, the MEK kinase signaling cascade leadsto increased PAX2 expression. In addition, the AT1R and AngIIupregulates PAX2 activation via JNK.

FIG. 27 shows schematic of blocking PAX2 expression as a therapy forprostate cancer. FIG. 27A shows PAX2 expression is regulated by the AT1Rsignaling pathway. Inhibition of PAX2 expression results in there-expression of DEFB1 and cancer cell death.

FIG. 27B shows compounds which block the AT1R, downstream kinases ordirectly suppresses PAX2 offer a novel approach to treating prostatecancer.

FIG. 28 shows comparison of DEFB1 and PAX2 expression with GleasonScore. DEFB1 relative expression levels were compared in benign clinicalsamples from 6 patients that underwent radical prostatectomies. HereGleason score inversely correlated with DEFB1 expression levels inadjacent benign prostate tissue. Patients with relative DEFB1 expressionlevels higher than 0.005 had Gleason sores of 6. However, those withexpression levels less than 0.005 had Gleason scores of 7.

FIG. 29 shows PAX2-DEFB1 ratio as a predictive factor for prostatecancer development. QRT-PCR was performed on laser capturemicrodissection (LCM) prostate tissue sections to determine relativeDEFB1 and PAX2 expression levels. DEFB1 expression levels decreased fromNormal to PIN to cancer. However, PAX2 expression increased from normalto PIN to cancer. In addition, patient #1457 with Gleason score 6 cancerhad more DEFB1 in normal tissue and PIN compared to patient #1569 withGleason score 7 cancer. Conversely, patient #1569 had higher PAX2 levelsin cancerous regions compared to patient #1457.

FIG. 30 shows the Donald Predictive Factor (DPF) is based on therelative PAX2-DEFB1 expression ratio. An increase in the DPF of prostatetissue increases the chance of developing prostate cancer. Tissue with aPAX2-DEFB1 ratio between 0 and 39 based on the DPF was normal (benign).Tissue with a PAX2-DEFB1 ratio between 40 and 99 represented PIN(pre-cancerous) based on the DPF scale. Finally, tissue with aPAX2-DEFB1 ratio between 100 and 500 was malignant (low to high gradecancer).

FIG. 31 shows analysis of hBD-1 expression in human prostate tissue.hBD-1 relative expression levels were compared in normal clinicalsamples from patients that underwent radical prostatectomies. The dashedline serves as a point of reference to compare values obtained betweengross and LCM-derived specimen, and corresponding Gleason scores areindicated above each bar. FIG. 31A shows hBD-1 expression levelscompared in tissues obtained by gross dissection. FIG. 31B shows hBD-1expression levels compared in tissue obtained by Laser CaptureMicrodissection.

FIG. 32 shows analysis of hBD-1 expression in prostate cell lines. FIG.32A shows hBD-1 expression levels compared relative to hPrEC cells inprostate cancer cell lines before and after hBD-1 induction. An asteriskrepresents statistically higher expression levels compared to hPrEC.Double asterisks represent statistically significant levels ofexpression compared to the cell line before hBD-1 induction (Student'st-test, p<0.05). FIG. 32B shows ectopic hBD-1 expression verified in theprostate cancer cell line DU145 by immunocytochemistry. hPrEC cells werestained for hBD-1 as appositive control (a: DIC and b: fluorescence).DU145 cells were transfected with hBD-1 and induced for 18 hours (c: DICand d: fluorescence). Sizebar=20 μM.

FIG. 33 shows analysis of hBD-1 cytotoxicity in prostate cancer cells.The prostate cell lines DU145, PC3, PC3/AR+ and LNCaP were treated withPon A to induce hBD-1 expression for 1-3 days after which MTT assay wasperformed to determine cell viability. Each bar represents themean±S.E.M. of three independent experiments performed in triplicate.

FIG. 34 shows QRT-PCR analysis of hBD-1 and cMYC expression in LCM humanprostate tissue sections of normal, PIN and tumor. Expression for eachgene is presented as expression ratios compared to β-actin. FIG. 35Ashows comparison of hBD-1 expression levels in normal, PIN and tumorsections. FIG. 35B shows comparison of cMYC expression level in normal,PIN and tumor sections.

FIG. 35 shows QRT-PCR analysis of hBD1 expression following PAX2knockdown with siRNA. hBD-1 expression levels are presented asexpression ratios compared to β-actin. An asterisk representsstatistically higher expression levels compared to the cell line beforePAX2 siRNA treatment (Student's t-test, p<0.05).

FIG. 36 shows silencing of PAX2 protein expression following PAX2 siRNAtreatment. FIG. 37A shows PAX2 expression examined by Western blotanalysis in HPrEC prostate primary cells (lane 1) and in DU145 (lane 2),PC3 (lane 3) and LNCaP (lane 4) prostate cancer cells. Blots werestripped and re-probed for -actin as an internal control to ensure equalloading. FIG. 37B shows Western blot analysis of DU145, PC3 and LNCaPall confirmed knockdown of PAX2 expression following transfection withPAX2 siRNA duplex. Again, blots were stripped and re-probed for β-actinas an internal control.

FIG. 37 shows analysis of prostate cancer cells growth after treatmentwith PAX2 siRNA. Phase contrast microscopic analysis of HPrEC (A), LNCaP(C), DU145 (E) and PC3 (G) at 6 days in the presence of negative controlnon-specific siRNA. There was a significant reduction in cell number inDU145 (D), PC3 (F) and LNCaP (H) following treatment with PAX2 siRNA.However, there appeared to be no effect in HPrEC (B). Bar=20 μm.

FIG. 38 shows analysis of cell death following siRNA silencing of PAX2.Prostate cancer cell lines PC3, DU145 and LNCaP were treated with PAX2siRNA or non-specific negative control siRNAs for 2, 4 or 6 days afterwhich MTT assay was performed. Knockdown of PAX2 resulted in a decreasein relative cell viability in all three lines. Results representmean±SD, n=9.

FIG. 39 shows analysis of caspase activity. DU145, PC3 and LNCaP cellswere stained with carboxyfluorescein-labeled fluoromethyl ketone todetected caspase activity following treatment with PAX2 siRNA. Analysisunder fluorescence revealed no caspase staining in control DU145 (A),PC3 cells (C) and LNCaP cells (E). However, cell treated with PAX2 siRNAinduced caspase activity in DU145 (B), PC3 (D) and LNCaP (F). Bar=20 μm.

FIG. 40 shows analysis of apoptotic factors following PAX2 siRNAtreatment. Changes in expression of pro-apoptotic factors were comparedin untreated control cells and in cells treated for 6 days with PAX2siRNA. FIG. 41A shows BAD expression increased in DU145, PC3 and LNCaPfollowing PAX2 knockdown. FIG. 41B shows BID expression levels increasedin LNCaP and DU145, but not in PC3 cells. FIG. 41C shows AKT expressiondecreased in LNCaP and DU145. However, there was no change in AKTexpression in PC3 cells following PAX2 knockdown. Results representmean±SD, n=9. Asterisks represents statistical differences (p<0.05).

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed method and compositions belong. It must be notedthat as used herein and in the appended claims, the singular forms “a,”“an,” and “the” include plural reference unless the context clearlydictates otherwise. Thus, for example, reference to “a peptide” includesa plurality of such peptides, reference to “the peptide” is a referenceto one or more peptides and equivalents thereof known to those skilledin the art, and so forth.

One aspect of the present invention relates to a method for monitoringcancer development. In certain embodiments, the method relates tomonitoring pre-cancerous conditions, such as intraepithelial neoplasia,and cancerous conditions in the prostate or breast of a subject. Themethod comprises determining a Paired Box 2 gene-to-beta defensin-1 gene(PAX2-to-DEFB1) expression ratio in cells obtained from the prostate orbreast of the subject, wherein the PAX2-to-DEFB1 expression ratio iscorrelated with prostate or breast conditions. The gene expression ratiomay be determined at the mRNA level (e.g., by RT-PCR or oligonucleotidearray) or at the protein level (e.g., by Western blot or antibodyarray).

In certain embodiments, the PAX2-to-DEFB1 expression ratio is determinedat the mRNA level and is referred to as the “Donald Predictive Factor”or “DPF” in the specification.

In certain embodiments, the PAX2-to-DEFB1 expression ratio (determinedat the RNA level) in prostate is used for distinguishing among normal,pre-cancerous and cancerous prostate conditions in the subject. In oneembodiment, a PAX2-to-DEFB1 ratio of less than 40:1 is indicative of anormal prostate condition, a PAX2-to-DEFB1 ratio of at least 40:1 toless than 100:1 is indicative of prostate intraepithelial neoplasia(PIN), and a PAX2-to-DEFB1 ratio of at least 100:1 is indicative ofprostate cancer.

Also provided is a method for diagnosing prostate cancer in a subject.The method comprises detecting in cells from the prostate of the subjectlevels of PAX2 and beta defensin-1 (DEFB1), wherein the ratio of PAX2 toDEFB1 is at least 100:1.

Also provided is a method of diagnosing prostate intraepithelialneoplasia (PIN) in a subject. The method comprises detecting in cellsfrom the prostate of the subject levels of PAX2 and beta DEFB1, whereinthe ratio of PAX2 to DEFB1 is at least 40:1 and less than 100:1.

In certain other embodiments, the PAX2-to-DEFB1 expression ratio (at theRNA level) in breast is used for distinguishing among non-cancerous(benign and/or pre-cancerous) and cancerous breast conditions in thesubject. In one embodiment, a PAX2-to-DEFB1 ratio less than 100:1 isindicative of non-cancerous (normal) and/or pre-cancerous (mammaryintraepithelial neoplasia (MIN)), and a PAX2-to-DEFB1 ratio of at least100:1 is indicative of breast cancer.

Also provided is a method for diagnosing breast cancer in a subject. Themethod comprises detecting in cells from the breast of the subjectlevels of PAX2 and DEFB1, wherein the ratio of PAX2 to DEFB1 of at least100:1 is indicative of breast cancer in the subject. Also provided is amethod of diagnosing non-cancerous breast conditions (normal and/or MIN)in a subject. The method comprises detecting in cells from the breast ofthe subject levels of PAX2 and DEFB1, wherein the ratio of PAX2 to DEFB1of less than 100:1 is indicative of non-cancerous breast conditions inthe subject.

In one embodiment, the method further comprising determining anoestrogen receptor/progesterone receptor (ER/PR) status in cellsobtained from the breast tissue with the breast condition. The ER/PRstatus of the breast tissue may be used, in combination with thePAX2-to-DEFB1 ratio in the same tissue, for determining the breastconditions in the subject.

As used hereinafter, the term “mammary intraepithelial neoplasia”includes lobular intraepithelial neoplasia and ductal intraepithelialneoplasia.

The monitoring and diagnosing methods of the present invention provideclinicians with a prognosticator for initiated or pre-cancerous tissue.Candidates for this test include patients at high risk (based on age,race) for cancer. As a diagnostic, a positive or negative PAX2 test canthen be followed by additional screening with biomarker to determinecancer site. In addition, these patients can be candidates for treatmentwith PAX2/DEFB1 modulators. Alternatively, this test can be used onpatients (i.e., those with triple negative breast cancer) as a measureof the effectiveness of their cancer therapy, to determine treatmentcourse, or to monitor cancer recurrence.

As another example, patients who present with potential indicators ofcancer such as the detection of nodules in the prostate during a digitalrectal exam by the clinician, or those who experience a sudden rise inPSA often are in the “Watchful Waiting” state. It is often difficult toascertain whether these patients have or will develop cancer. Thedetection of PAX2-to-DEFB1 ratio in samples, such as plasma/serum, fromthese patients can be used to assist the decision to obtain a biopsy inmen with suspected prostate cancer, which can lead to a reduction in thenumber of unnecessary prostatic biopsies and earlier intervention fortheir disease.

Prostate Cancer

Prostate cancer screening currently consists of a rectal examination andmeasurement of prostate specific antigen (PSA) levels. These methodslack specificity as digital rectal examination has considerableinter-examiner variability and PSA levels may be elevated in benignprostatic hyperplasia (BPH), prostatic inflammation and otherconditions.

Prostate cancers can be scored using the Gleason system (Gleason, et al.1966). This uses tissue architecture rather than cytological features. Agrade of 1 to 5 (well to poorly differentiated) is used, and thecombined score of the most frequent and more severe areas of the lesionare combined. Gleason scores provide prognostic information that may bevaluable in addition to the assessment of the stage of the tumor(staging). Gleason scores of 2 to 4 and 8 to have good predictive value,but about three quarters of tumors have intermediate values.

Two principal systems are used for staging prostate cancer: TNM and theJewett system (Benson & Olsson, et al. 1989). Staging takes in toaccount any metastatic spread of the tumor and is difficult, because itis difficult to assess either local lymph node involvement or localinvasion. Tumor size is also difficult to measure as tumor tissue cannotbe distinguished macroscopically from normal prostate tissue, andbecause the prostate gland lacks a distinct capsule and is surrounded bya layer of fibrous fatty tissue.

Four categories describe the prostate tumor's (T) stage, ranging from T1to T4. For T1, the cancer is microscopic, unilateral and non palpable.The doctor can't feel the tumor or see it with imaging such astransrectal ultrasound. Treatment for BPH may have disclosed thedisease, or it was confirmed through the use of a needle biopsy donebecause of an elevated PSA. For T2, the doctor can feel the cancer witha DRE. It appears the disease is confined to the prostate gland on oneor both sides of the gland. For T3, the cancer has advanced to tissueimmediately outside the gland. For T4, the cancer has spread to otherparts of the body.

Present screening methods are therefore unsatisfactory; there is noreliable method for diagnosing prostate cancer, or predicting orpreventing its possible metastatic spread, which is the main cause ofdeath for most patients.

Breast Cancer

The commonly used screening methods for breast cancer include self andclinical breast exams, x-ray mammography, and breast Magnetic ResonanceImaging (MRI). The most recent technology for breast cancer screening isultrasound computed tomography, which uses sound waves to create athree-dimensional image and detect breast cancer without the use ofdangerous radiation used in x-ray mammography. Genetic testing may alsobe used. Genetic testing for breast cancer typically involves testingfor mutations in the BRCA genes. It is not a generally recommendedtechnique except for those at elevated risk for breast cancer.

The incidence of breast cancer, a leading cause of death in women, hasbeen gradually increasing in the United States over the last thirtyyears. While the pathogenesis of breast cancer is unclear,transformation of normal breast epithelium to a malignant phenotype maybe the result of genetic factors, especially in women under 30 (Miki etal., 1994, Science, 266:66-71). The discovery and characterization ofBRCA1 and BRCA2 has recently expanded our knowledge of genetic factorswhich can contribute to familial breast cancer. Germ-line mutationswithin these two loci are associated with a 50 to 85% lifetime risk ofbreast and/or ovarian cancer (Casey, 1997, Curr. Opin. Oncol. 9:88-93;Marcus et al, 1996, Cancer 77:697-709). However, it is likely thatother, non-genetic factors also have a significant effect on theetiology of the disease. Regardless of its origin, breast cancermorbidity and mortality increases significantly if it is not detectedearly in its progression. Thus, considerable effort has focused on theearly detection of cellular transformation and tumor formation in breasttissue.

Currently, the principal manner of identifying breast cancer is throughdetection of the presence of dense tumorous tissue. This may beaccomplished to varying degrees of effectiveness by direct examinationof the outside of the breast, or through mammography or other X-rayimaging methods (Jatoi, 1999, Am. J. Surg. 177:518-524). The latterapproach is not without considerable cost, however. Every time amammogram is taken, the patient incurs a small risk of having a breasttumor induced by the ionizing properties of the radiation used duringthe test. In addition, the process is expensive and the subjectiveinterpretations of a technician can lead to imprecision, e.g., one studyshowed major clinical disagreements for about one-third of a set ofmammograms that were interpreted individually by a surveyed group ofradiologists. Moreover, many women find that undergoing a mammogram is apainful experience. Accordingly, the National Cancer Institute has notrecommended mammograms for women under fifty years of age, since thisgroup is not as likely to develop breast cancers as are older women. Itis compelling to note, however, that while only about 22% of breastcancers occur in women under fifty, data suggests that breast cancer ismore aggressive in pre-menopausal women.

PAX2

PAX genes are a family of nine developmental control genes coding fornuclear transcription factors. They play an important role inembryogenesis and are expressed in a very ordered temporal and spatialpattern. They all contain a “paired box” region of 384 base pairsencoding a DNA binding domain which is highly conserved throughoutevolution (Stuart, E T, et al. 1994). The influence of Pax genes ondevelopmental processes has been demonstrated by the numerous naturalmouse and human syndromes that can be attributed directly to even aheterozygous insufficiency in a Pax gene. A PAX2 sequence is given inDressler, et al. 1990. The amino acid sequences of the human PAX2protein and its variants, as well as the DNA sequences encoding theproteins, are listed in SEQ ID NOS: 58-69 (SEQ ID NO:58, amino acidsequence encoded by exon 1 of the human PAX2 gene; SEQ ID NO:59, humanPAX2 gene promoter and exon 1; SEQ ID NO:60, amino acid sequence of thehuman PAX2; SEQ ID NO:61, human PAX2 gene; SEQ ID NO:62, amino acidsequence of the human PAX2 gene variant b; SEQ ID NO:63, human PAX2 genevariant b; SEQ ID NO:64, amino acid sequence of the human PAX2 genevariant c; SEQ ID NO:65, human PAX2 gene variant c; SEQ ID NO:66, aminoacid sequence of the human PAX2 gene variant d; SEQ ID NO:67, human PAX2gene variant d; SEQ ID NO:68, amino acid sequence of the human PAX2 genevariant e; SEQ ID NO:69 human PAX2 gene variant e).

Examples of cancers in which PAX2 expression has been detected arelisted in Table 1

TABLE 1 PAX2-expressing cancers Estimated Estimated Estimated EstimatedPAX2 Expressing New Cases Deaths New Cases Deaths Cancers in US in USGlobal Global Prostate 234,460 27,350 679,023 221,002 Breast 214,60041,430 1,151,298 410,712 Ovarian 20,180 15,310 204,500 124,860 Renal38,890 12,840 208,479 101,895 Brain 12,820 18,820 189,485 141,650Cervical 9,710 3,700 493,243 273,505 Bladder 61,420 13,060 356,556145,009 Leukemia 35,020 22,280 300,522 222,506 Kaposi Sarcoma Data NotData Not Data Not Data Not Available Available Available AvailableTOTAL(approx.) 627,100 154,790 3,583,106 1,641,139

DEFB1

Beta-defensins are cationic peptides with broad-spectrum antimicrobialactivity that are products of epithelia and leukocytes. These two exon,single gene products are expressed at epithelial surfaces and secretedat sites including the skin, cornea, tongue, gingiva, salivary glands,esophagus, intestine, kidney, urogenital tract, and the respiratoryepithelium. To date, five beta-defensin genes of epithelial origin havebeen identified and characterized in humans: DEFB1 (Bensch et al.,1995), DEFB 2 (Harder et al., 1997), DEFB3 (Harder et al., 2001; Jia etal., 2001), DEFB4, and HE2/EP2.

The primary structure of each beta-defensin gene product ischaracterized by small size, a six cysteine motif, high cationic chargeand exquisite diversity beyond these features. The most characteristicfeature of defensin proteins is their six-cysteine motif that forms anetwork of three disulfide bonds. The three disulfide bonds in thebeta-defensin proteins are between C1-C5, C2-C4 and C3-C6. The mostcommon spacing between adjacent cysteine residues is 6, 4, 9, 6, 0. Thespacing between the cysteines in the beta-defensin proteins can vary byone or two amino acids except for C5 and C6, located nearest the carboxyterminus. In all known vertebrate beta-defensin genes, these twocysteine residues are adjacent to each other.

A second feature of the beta-defensin proteins is their small size. Eachbeta-defensin gene encodes a preproprotein that ranges in size from 59to 80 amino acids with an average size of 65 amino acids. This geneproduct is then cleaved by an unknown mechanism to create the maturepeptide that ranges in size from 36 to 47 amino acids with an averagesize of 45 amino acids. The exceptions to these ranges are the EP2/HE2gene products that contain the beta-defensin motif and are expressed inthe epididymis.

A third feature of beta-defensin proteins is the high concentration ofcationic residues. The number of positively charged residues (arginine,lysine, histidine) in the mature peptide ranges from 6 to 14 with anaverage of 9.

The final feature of the beta-defensin gene products is their diverseprimary structure but apparent conservation of tertiary structure.Beyond the six cysteines, no single amino acid at a given position isconserved in all known members of this protein family. However, thereare positions that are conserved that appear to be important forsecondary and tertiary structures and function.

Despite the great diversity of the primary amino acid sequence of thebeta-defensin proteins, the limited data suggests that the tertiarystructure of this protein family is conserved. The structural core is atriple-stranded, antiparallel beta-sheet, as exemplified for theproteins encoded by BNBD-12 and DEFB2. The three beta-strands areconnected by a beta-turn, and an alpha-hairpin loop, and the secondbeta-strand also contains a beta-bulge. When these structures are foldedinto their proper tertiary structure, the apparently random sequences ofcationic and hydrophobic residues are concentrated into two faces of aglobular protein. One face is hydrophilic and contains many of thepositively charged side chains and the other is hydrophobic. Insolution, the HBD-2 protein encoded by the DEFB2 gene exhibited analpha-helical segment near the N-terminus not previously ascribed tosolution structures of alpha-defensins or to the beta-defensin BNBD-12.The amino acids whose side chains are directed toward the surface of theprotein are less conserved between beta defensin proteins while theamino acid residues in the three beta-strands of the core beta-sheet aremore highly conserved.

Beta-defensin peptides are produced as pre-pro-peptides and then cleavedto release a C-terminal active peptide fragment; however the pathwaysfor the intracellular processing, storage and release of the humanbeta-defensin peptides in airway epithelia are unknown.

Determination of PAX2-to-DEFB1 Expression Ratio

Levels of PAX2 and DEFB1 expression in a tissue can be measured anymethod known in the art. In certain embodiments, the levels of PAX2 andDEFB1 expression in a target tissue are determined by determining thelevels of PAX2 and DEFB1 in a cell or cells obtained directly from thetarget tissue, such as a biopsy sample. In other embodiments, the levelsof PAX2 and DEFB1 expression in a target tissue are determinedindirectly by determining the levels of PAX2 and DEFB1 in certain bodyfluids such as blood or plasma.

In certain embodiments, levels of PAX2 and DEFB1 expression in theprostate or breast of the subject can be measured using a cell samplefrom the prostate or breast. Cell samples include biopsy samples of theprostate or breast and blood sample. Biopsy is a procedure in whichsmall tissue samples are removed from a target organ for furtheranalysis. Prostate biopsy is typically performed when the scores from aPSA blood test rise to a level that is associated with the possiblepresence of prostate cancer. Similarly, breast biopsy is typicallyperformed in patients with breast lumps or suspicious mammograms.

Levels of gene expression can be evaluated at the RNA and proteinlevels. The RNA levels may be measured, for example, with DNA arrays,RT-PCR and Northern Blotting. The protein levels may be measured withimmunoassays and enzyme assays. In certain embodiments, thePAX2-to-DEFB1 expression ratio is determined by determining theexpression level of PAX2 gene relative to the expression level of acontrol gene, determining the expression level of DEFB1 gene relative tothe expression level of the same control gene, and calculating thePAX2-to-DEFB1 expression ratio based on the expression levels of PAX2and DEFB1. In one embodiment, the control gene is the glyceraldehyde3-phosphate dehydrogenase (GAPDH) gene.

Oligonucleotide Microarray

An oligonucleotide microarray consists of an arrayed series of aplurality of microscopic spots of oligonucleotides, called features,each containing a small amount (typically in the range of picomoles) ofa specific oligonucleotide sequence. The specific oligonucleotidesequence can be a short section of a gene or other oligonucleotideelement that are used as probes to hybridize a cDNA or cRNA sample underhigh-stringency conditions. Probe-target hybridization is usuallydetected and quantified by fluorescence-based detection offluorophore-labeled targets to determine relative abundance of nucleicacid sequences in the target.

The probes are typically attached to a solid surface by a covalent bondto a chemical matrix (via epoxy-silane, amino-silane, lysine,polyacrylamide or others). The solid surface can be glass or a siliconchip or microscopic beads. Oligonucleotide arrays are different fromother types of microarray only in that they either measure nucleotidesor use oligonucleotide as part of its detection system.

To detect gene expression in target tissue or cells using anoligonucleotide array, nucleic acid of interest is purified from thetarget tissue or cells. The nucleotide can be all RNA for expressionprofiling, DNA for comparative hybridization, or DNA/RNA bound to aparticular protein which is immunoprecipitated (ChIP-on-chip) forepigenetic or regulation studies.

In one embodiment, total RNA is isolated (total as it is nuclear andcytoplasmic) by guanidinium thiocyanate-phenol-chloroform extraction(e.g. Trizol). The purified RNA may be analyzed for quality (e.g., bycapillary electrophoresis) and quantity (e.g., by using a nanodropspectrometer. The total RNA is RNA is reverse transcribed into DNA witheither polyT primers or random primers. The DNA products may beoptionally amplified by PCR. A label is added to the amplificationproduct either in the RT step or in an additional step afteramplification if present. The label can be a fluorescent label orradioactive labels. The labeled DNA products are then hybridized to themicroarray. The microarray is then washed and scanned. The expressionlevel of the gene of interest is determined based on the hybridizationresult using method well known in the art.

Immunoassays

Immunoassays, in their most simple and direct sense, are binding assaysinvolving binding between antibodies and antigen. Many types and formatsof immunoassays are known and all are suitable for detecting thedisclosed biomarkers. Examples of immunoassays are enzyme linkedimmunosorbent assays (ELISAs), radioimmunoassays (RIA), radioimmuneprecipitation assays (RIPA), immunobead capture assays, Westernblotting, dot blotting, gel-shift assays, Flow cytometry, proteinarrays, multiplexed bead arrays, magnetic capture, in vivo imaging,fluorescence resonance energy transfer (FRET), and fluorescencerecovery/localization after photobleaching (FRAP/FLAP).

In general, immunoassays involve contacting a sample suspected ofcontaining a molecule of interest (such as the disclosed biomarkers)with an antibody to the molecule of interest or contacting an antibodyto a molecule of interest (such as antibodies to the disclosedbiomarkers) with a molecule that can be bound by the antibody, as thecase may be, under conditions effective to allow the formation ofimmunocomplexes. In many forms of immunoassay, the sample-antibodycomposition, such as a tissue section, ELISA plate, dot blot or Westernblot, can then be washed to remove any non-specifically bound antibodyspecies, allowing only those antibodies specifically bound within theprimary immune complexes to be detected.

Radioimmune Precipitation Assay (RIPA) is a sensitive assay usingradiolabeled antigens to detect specific antibodies in serum. Theantigens are allowed to react with the serum and then precipitated usinga special reagent such as, for example, protein A sepharose beads. Thebound radiolabeled immunoprecipitate is then commonly analyzed by gelelectrophoresis. Radioimmunoprecipitation assay (RIPA) is often used asa confirmatory test for diagnosing the presence of HIV antibodies. RIPAis also referred to in the art as Farr Assay, Precipitin Assay,Radioimmune Precipitin Assay; Radioimmunoprecipitation Analysis;Radioimmunoprecipitation Analysis, and RadioimmunoprecipitationAnalysis.

Also contemplated are immunoassays wherein the protein or antibodyspecific for the protein is bound to a solid support (e.g., tube, well,bead, or cell) to capture the antibody or protein of interest,respectively, from a sample, combined with a method of detecting theprotein or antibody specific for the protein on the support. Examples ofsuch immunoassays include Radioimmunoassay (RIA), Enzyme-LinkedImmunosorbent Assay (ELISA), Flow cytometry, protein array, multiplexedbead assay, and magnetic capture.

Protein arrays are solid-phase ligand binding assay systems usingimmobilized proteins on surfaces which include glass, membranes,microtiter wells, mass spectrometer plates, and beads or otherparticles. The assays are highly parallel (multiplexed) and oftenminiaturized (microarrays, protein chips). Their advantages includebeing rapid and automatable, capable of high sensitivity, economical onreagents, and giving an abundance of data for a single experiment.Bioinformatics support is important; the data handling demandssophisticated software and data comparison analysis. However, thesoftware can be adapted from that used for DNA arrays, as can much ofthe hardware and detection systems.

Capture arrays form the basis of diagnostic chips and arrays forexpression profiling. They employ high affinity capture reagents, suchas conventional antibodies, single domains, engineered scaffolds,peptides or nucleic acid aptamers, to bind and detect specific targetligands in high throughput manner. Antibody arrays are availablecommercially. In addition to the conventional antibodies, Fab and scFvfragments, single V-domains from camelids or engineered humanequivalents (Domantis, Waltham, Mass.) may also be useful in arrays.

Nonprotein capture molecules, notably the single-stranded nucleic acidaptamers which bind protein ligands with high specificity and affinity,are also used in arrays (SomaLogic, Boulder, Colo.). Aptamers areselected from libraries of oligonucleotides by the Selex™ procedure andtheir interaction with protein can be enhanced by covalent attachment,through incorporation of brominated deoxyuridine and UV-activatedcrosslinking (photoaptamers). Photocrosslinking to ligand reduces thecrossreactivity of aptamers due to the specific steric requirements.Aptamers have the advantages of ease of production by automatedoligonucleotide synthesis and the stability and robustness of DNA; onphotoaptamer arrays, universal fluorescent protein stains can be used todetect binding.

An alternative to an array of capture molecules is one made through‘molecular imprinting’ technology, in which peptides (e.g., from theC-terminal regions of proteins) are used as templates to generatestructurally complementary, sequence-specific cavities in apolymerizable matrix; the cavities can then specifically capture(denatured) proteins that have the appropriate primary amino acidsequence (ProteinPrint™, Aspira Biosystems, Burlingame, Calif.).

Another methodology which can be used diagnostically and in expressionprofiling is the ProteinChip® array (Ciphergen, Fremont, Calif.), inwhich solid phase chromatographic surfaces bind proteins with similarcharacteristics of charge or hydrophobicity from mixtures such as plasmaor tumor extracts, and SELDI-TOF mass spectrometry is used to detectionthe retained proteins.

Other useful methodology includes large-scale functional chipsconstructed by immobilizing large numbers of purified proteins on achip, and multiplexed bead assays.

Antibodies

The term “antibodies” is used herein in a broad sense and includes bothpolyclonal and monoclonal antibodies. In addition to intactimmunoglobulin molecules, also included in the term “antibodies” arefragments or polymers of those immunoglobulin molecules, and human orhumanized versions of immunoglobulin molecules or fragments thereof, aslong as they are chosen for their ability to interact with, for example,PAX2 or DEFB1, such that PAX2 is inhibited from interacting with DEFB1.Antibodies that bind the disclosed regions of PAX2 or DEFB1 involved inthe interaction between PAX2 and DEFB1 are also disclosed. Theantibodies can be tested for their desired activity using the in vitroassays described herein, or by analogous methods, after which their invivo therapeutic and/or prophylactic activities are tested according toknown clinical testing methods.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, as long as they exhibit thedesired antagonistic activity (See, U.S. Pat. No. 4,816,567 and Morrisonet al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

As used herein, the term “antibody” or “antibodies” can also refer to ahuman antibody and/or a humanized antibody. Many non-human antibodies(e.g., those derived from mice, rats, or rabbits) are naturallyantigenic in humans, and thus can give rise to undesirable immuneresponses when administered to humans. Therefore, the use of human orhumanized antibodies in the methods serves to lessen the chance that anantibody administered to a human will evoke an undesirable immuneresponse. Methods for humanizing non-human antibodies are well known inthe art.

Pharmacogenomics

In another embodiment, the PAX2 and/or DEFB1 expression profiles areused for determine pharmacogenomics of breast cancer. Pharmacogenomicsrefers to the relationship between an individual's genotype and thatindividual's response to a foreign compound or drug. Differences inmetabolism of therapeutics can lead to severe toxicity or therapeuticfailure by altering the relation between dose and blood concentration ofthe pharmacologically active drug. Thus, a physician or clinician mayconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer an anti-cancer drug, aswell as tailoring the dosage and/or therapeutic regimen of treatmentwith the anti-cancer drug.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. In general, two types of pharmacogeneticconditions can be differentiated. Genetic conditions transmitted as asingle factor altering the way drugs act on the body (altered drugaction) or genetic conditions transmitted as single factors altering theway the body acts on drugs (altered drug metabolism). Thesepharmacogenetic conditions can occur either as rare genetic defects oras naturally-occurring polymorphisms. For example, glucose-6-phosphatedehydrogenase deficiency (G6PD) is a common inherited enzymopathy inwhich the main clinical complication is hemolysis after ingestion ofoxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans)and consumption of fava beans.

One pharmacogenomics approach to identifying genes that predict drugresponse, known as “a genome-wide association,” relies primarily on ahigh-resolution map of the human genome consisting of already knowngene-related sites (e.g., a “bi-allelic” gene marker map which consistsof 60,000-100,000 polymorphic or variable sites on the human genome,each of which has two variants). Such a high-resolution genetic map canbe compared to a map of the genome of each of a statisticallysubstantial number of subjects taking part in a Phase II/III drug trialto identify genes associated with a particular observed drug response orside effect. Alternatively, such a high resolution map can be generatedfrom a combination of some ten-million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, an “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, an SNP may occur once per every 1,000 bases of DNA.An SNP may be involved in a disease process. However, the vast majorityof SNPs may not be disease associated. Given a genetic map based on theoccurrence of such SNPs, individuals can be grouped into geneticcategories depending on a particular pattern of SNPs in their individualgenome. In such a manner, treatment regimens can be tailored to groupsof genetically similar individuals, taking into account traits that maybe common among such genetically similar individuals. Thus, mapping ofthe PAX2 and/or DEFB1 to SNP maps of breast patients may allow easieridentification of these genes according to the genetic methods describedherein.

Alternatively, a method termed the “candidate gene approach,” can beutilized to identify genes that predict drug response. According to thismethod, if a gene that encodes a drug target is known, all commonvariants of that gene can be fairly easily identified in the populationand it can be determined if having one version of the gene versusanother is associated with a particular drug response.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYPZC19) has provided an explanation as to why some subjectsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer and poor metabolizer. Theprevalence of poor metabolizer phenotypes is different among differentpopulations. For example, the gene coding for CYP2D6 is highlypolymorphic and several mutations have been identified in poormetabolizers, which all lead to the absence of functional CYP2D6. Poormetabolizers of CYP2D6 and CYP2C19 quite frequently experienceexaggerated drug response and side effects when they receive standarddoses. If a metabolite is the active therapeutic moiety, poormetabolizers show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Alternatively, a method termed the “gene expression profiling” can beutilized to identify genes that predict drug response. For example, thegene expression of an animal dosed with a drug can give an indicationwhether gene pathways related to toxicity have been turned on.

Information generated from more than one of the above pharmacogenomicsapproaches can be used to determine appropriate dosage and treatmentregimens for prophylactic or therapeutic treatment an individual. Thisknowledge, when applied to dosing or drug selection, can avoid adversereactions or therapeutic failure and thus enhance therapeutic orprophylactic efficiency when treating a subject with a breast condition.

In one embodiment, the PAX2 and/or DEFB1 expression profiles, as well asthe ER/PR status, in a subject are used to determine the appropriatetreatment regimens for an individual with a breast condition.

In another embodiment, the PAX2 expression level (typically determine inreference to a control gene as actin gene or GAPDH gene) is used inpatients with triple negative breast cancer (i.e., oestrogen receptor(ER) negative, progesterone receptor (PR) negative, human epidermalgrowth factor receptor 2 (HER2) negative) to measure of theeffectiveness of cancer therapy, to determine treatment course, or tomonitor cancer recurrence.

Diagnosis Kits

Another aspect of the present invention relates to a kit for monitoringbreast conditions. In one embodiment, the kit for monitoring breastconditions comprises: one or more pairs of oligonucleotide primers fordetecting PAX2 expression in a tissue sample, one or more pairs ofoligonucleotide primers for detecting DEFB1 expression in the tissuesample, and instructions on how to determine the PAX2-to-DEFB1expression ratio in a tissue sample using the primers. In anotherembodiment, the one or more pairs of oligonucleotide primers fordetecting PAX2 expression comprising an oligonucleotide primer pairselected from the group consisting of SEQ ID NOS: 43 and 47, SEQ ID NOS:44 and 48, and SEQ ID NOS: 45 and 49. In another embodiment, the one ormore pairs of oligonucleotide primers for detecting DEFB1 expressioncomprising SEQ ID NOS: 35 and 37.

In another embodiment, the kit further comprises one or more pairs ofcontrol oligonucleotide primers. In one embodiment, the one or morepairs of control oligonucleotide primers comprise oligonucleotideprimers for detecting expression of β-actin expression. In a preferredembodiment, the oligonucleotide primers for detecting expression ofβ-actin expression comprise SEQ ID NOS: 34 and 36.

In another embodiment, the one or more pairs of control oligonucleotideprimers comprise oligonucleotide primers for detecting expression ofGAPDH expression.

In a preferred embodiment, the oligonucleotide primers for detectingexpression of GAPDH expression comprise SEQ ID NOS: 42 and 46.

In another related embodiment, the kit further comprises one or morereagents for PCR reaction.

In yet another related embodiment, the kit further comprises one or morereagents for RNA extraction.

In another embodiment, the kit for monitoring breast conditionscomprises an oligonucleotide microarray having oligonucleotide probesfor detecting PAX2 and DEFB1 expression and instructions on how todetermine the PAX2-to-DEFB1 expression ratio in a tissue sample usingthe oligonucleotide microarray.

In a related embodiment, the kit further comprises reagents forextracting RNA from a tissue sample.

The present invention is further illustrated by the following exampleswhich should not be construed as limiting. The contents of allreferences, patents and published patent applications cited throughoutthis application, as well as the Figures and Tables are incorporatedherein by reference.

Example 1 Human Beta Defensin-1 is Cytotoxic to Late-Stage ProstateCancer and Plays a Role in Prostate Cancer Tumor Immunity

In this example, DEFB1 was cloned into an inducible expression system toexamine what effect it had on normal prostate epithelial cells, as wellas androgen receptor positive (AR+) and androgen receptor negative (AR−)prostate cancer cell lines. Induction of DEFB1 expression resulted in adecrease in cellular growth in AR− cells DU145 and PC3, but had noeffect on the growth of the AR+ prostate cancer cells LNCaP. DEFB1 alsocaused rapid induction of caspase-mediated apoptosis. Data presentedhere are the first to provide evidence of its role in innate tumorimmunity and indicate that its loss contributes to tumor progression inprostate cancer.

Materials and Methods

Cell Lines: The cell lines DU145 were cultured in DMEM medium, PC3 weregrown in F12 medium, and LNCaP were grown in RPMI medium (LifeTechnologies, Inc., Grand Island, N.Y.). Growth media for all threelines was supplemented with 10% (v/v) fetal bovine serum (LifeTechnologies). The hPrEC cells were cultured in prostate epitheliumbasal media (Cambrex Bio Science, Inc., Walkersville, Md.). All celllines were maintained at 37° C. and 5% CO2.

Tissue Samples and Laser Capture Microdissection: Prostate tissuesobtained from consented patients that underwent radical prostatectomywere acquired through the Hollings Cancer Center tumor bank inaccordance with an Institutional Review Board-approved protocol. Thisincluded guidelines for the processing, sectioning, histologicalcharacterization, RNA purification and PCR amplification of samples.Following pathologic examination of frozen tissue sections, lasercapture microdissection (LCM) was performed to ensure that the tissuesamples assayed consisted of pure populations of benign prostate cells.For each tissue section analyzed, LCM was performed at three differentregions containing benign tissue and the cells collected were thenpooled.

Cloning of DEFB1 Gene: DEFB1 cDNA was generated from RNA by reversetranscription-PCR. The PCR primers were designed to contain ClaI andKpnI restriction sites. DEFB1 PCR products were restriction digestedwith ClaI and KpnI and ligated into a TA cloning vector. The TA/DEFB1vector was then transfected into E. coli by heat shock and individualclones were selected and expanded. Plasmids were isolated by CellCulture DNA Midiprep (Qiagen, Valencia, Calif.) and sequence integrityverified by automated sequencing. The DEFB1 gene fragment was thenligated into the pTRE2 digested with ClaI and KpnI, which served as anintermediate vector for orientation purposes. Then the pTRE2/DEFB1construct was digested with ApaI and KpnI to excise the DEFB1 insert,which was ligated into pIND vector of the Ecdysone Inducible ExpressionSystem (Invitrogen, Carlsbad, Calif.) also double digested with ApaI andKpnI. The construct was again transfected into E. coli and individualclones were selected and expanded. Plasmids were isolated and sequenceintegrity of pIND/DEFB1 was again verified by automated sequencing.

Transfection: Cells (1×10⁶) were seeded onto 100-mm Petri dishes andgrown overnight. Then the cells were co-transfected using Lipofectamine2000 (Invitrogen, Carlsbad, Calif.) with 1 μg of pVgRXR plasmid, whichexpresses the heterodimeric ecdysone receptor, and 1 μg of thepIND/DEFB1 vector construct or empty pIND control vector in Opti-MEMmedia (Life Technologies, Inc., Grand Island, N.Y.).

RNA Isolation and Quantitative RT-PCR: In order to verify DEFB1 proteinexpression in the cells transfected with DEFB1 construct, RNA wascollected after a 24 hour induction period with Ponasterone A (Pon A).Briefly, total RNA was isolated using the SV Total RNA Isolation System(Promega, Madison, Wis.) from approximately 1×10⁶ cells harvested bytrypsinizing. Here, cells were lysed and total RNA was isolated bycentrifugation through spin columns. For cells collected by LCM, totalRNA was isolated using the PicoPure RNA Isolation Kit (ArcturusBiosciences, Mt. View, Calif.) following the manufacturer's protocol.Total RNA (0.5 μg per reaction) from both sources was reversetranscribed into cDNA utilizing random primers (Promega). AMV ReverseTranscriptase II enzyme (500 units per reaction; Promega) was used forfirst strand synthesis and Tfl DNA Polymerase for second strandsynthesis (500 units per reaction; Promega) as per the manufacturer'sprotocol. In each case, 50 pg of cDNA was used per ensuing PCR reaction.Two-step QRT-PCR was performed on cDNA generated using the MultiScribeReverse Transcripatase from the TaqMan Reverse Transcription System andthe SYBR® Green PCR Master Mix (Applied Biosystems).

The primer pair for DEFB1 (Table 2) was generated from the publishedDEFB1 sequence (GenBank Accession No. U50930). Forty cycles of PCR wereperformed under standard conditions using an annealing temperature of56° C. In addition, β-actin (Table 3) was amplified as a housekeepinggene to normalize the initial content of total cDNA. DEFB1 expressionwas calculated as the relative expression ratio between DEFB1 andβ-actin and was compared in cells lines induced and uninduced for DEFB1expression, as well as LCM benign prostatic tissue. As a negativecontrol, QRT-PCR reactions without cDNA template were also performed.All reactions were run three times in triplicate.

TABLE 2 Sequences of QRT-PCR Primers. Sense (5′-3′) β-actin5′-CCTGGCACCCAGCACAAT-3′ SEQ ID NO: 34 DEFB1 5′-GTTGCCTGCCAGTCGCCATGASEQ ID GAACTTCCTAC-3′ NO: 35 Antisense (5′-3′) β-actin5′-GCCGATCCACACGGAGTACT-3′ SEQ ID NO: 36 DEFB1 5′-TGGCCTTCCCTCTGTAACAGGTSEQ ID GCCTTGAATT-3′ NO: 37

MTT Cell Viability Assay: To examine the effects of DEFB1 on cellgrowth, metabolic 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazoliumbromide (MTT) assays were performed. PC3, DU145 and LNCaP cellsco-transfected with pVgRXR plasmid and pIND/DEFB1 construct or emptypIND vector were seeded onto a 96-well plate at 1-5×10³ cells per well.Twenty-four hours after seeding, fresh growth medium was addedcontaining 10 μM Ponasterone A daily to induce DEFB1 expression for 24-,48- and 72 hours after which the MTT assay was performed according tothe manufacturer's instructions (Promega). Reactions were performedthree times in triplicate.

Flow Cytometry: PC3 and DU145 cells co-transfected with the DEFB1expression system were grown in 60-mm dishes and induced for 12, 24, and48 hours with 10 μM Ponasterone A. Following each incubation period, themedium was collected from the plates (to retain any detached cells) andcombined with PBS used to wash the plates. The remaining attached cellswere harvested by trypsinization and combined with the detached cellsand PBS. The cells were then pelleted at 4° C. (500×g) for 5 min, washedtwice in PBS, and resuspended in 100 μl of 1× Annexin binding buffer(0.1 M Hepes/NaOH at pH 7.4, 1.4 M NaCl, 25 mM CaCl2) containing 5 μl ofAnnexin V-FITC and 5 μl of PI. The cells were incubated at RT for min inthe dark, then diluted with 4001 of 1× Annexin binding buffer andanalyzed by FACscan (Becton Dickinson, San Jose, Calif.). All reactionswere performed three times.

Microscopic Analysis: Cell morphology was analyzed by phase contrastmicroscopy. DU145, PC3 and LNCaP cells containing no vector, emptyplasmid or DEFB1 plasmid were seeded onto 6 well culture plates (BDFalcon, USA). The following day plasmid-containing cells were inducedfor a period of 48 h with media containing 10 μM Ponasterone A, whilecontrol cells received fresh media. The cells were then viewed under aninverted Zeiss IM 35 microscope (Carl Zeiss, Germany). Phase contrastpictures of a field of cells were obtained using the SPOT Insight Mosaic4.2 camera (Diagnostic Instruments, USA). Cells were examined by phasecontrast microscopy under 32× magnification and digital images werestored as uncompressed TIFF files and exported into Photoshop CSsoftware (Adobe Systems, San Jose, Calif.) for image processing and hardcopy presentation.

Caspase Detection: Detection of caspase activity in the prostate cancercell lines was performed using APO LOGIX™ Carboxyfluorescin Caspasedetection kit (Cell Technology, Mountain View, Calif.). Active caspaseswere detected through the use of a FAM-VAD-FMK inhibitor thatirreversibly binds to active caspases. Briefly, DU145 and PC3 cells(1.5-3×10⁵) containing the DEFB1 expression system were plated in 35 mmglass bottom microwell dishes (Matek, Ashland, Mass.) and treated for 24hours with media only or with media containing PonA as previouslydescribed. Next, 101 of a 30× working dilution of carboxyfluoresceinlabeled peptide fluoromethyl ketone (FAM-VAD-FMK) was added to 300 μl ofmedia and added to each 35 mm dish. Cells were then incubated for 1 hourat 37° C. under 5% CO2. Then, the medium was aspirated and the cellswere washed twice with 2 ml of a 1× Working dilution Wash Buffer. Cellswere viewed under differential interference contrast (DIC) or underlaser excitation at 488 nm. The fluorescent signal was analyzed using aconfocal microscope (Zeiss LSM 5 Pascal) and a 63×DIC oil lens with aVario 2 RGB Laser Scanning Module.

Statistical Analysis: Statistical differences were evaluated using theStudent's t-test for unpaired values. P values were determined by atwo-sided calculation, and a P value of less than 0.05 was consideredstatistically significant.

DEFB1 Expression in Prostate Tissue and Cell Lines: DEFB1 expressionlevels were measured by QRT-PCR in benign and malignant prostatictissue, hPrEC prostate epithelial cells and DU145, PC3 and LNCaPprostate cancer cells. DEFB1 expression was detected in all of thebenign clinical samples. The average amount of DEFB1 relative expressionwas 0.0073. In addition, DEFB1 relative expression in hPrEC cells was0.0089. There was no statistical difference in DEFB1 expression detectedin the benign prostatic tissue samples and hPrEC (FIG. 1A). Analysis ofthe relative DEFB1 expression levels in the prostate cancer cell linesrevealed significantly lower levels in DU145, PC3 and LNCaP. As afurther point of reference, relative DEFB1 expression was measured inthe adjacent malignant section of prostatic tissue from patient #1215.There were no significant differences in the level of DEFB1 expressionobserved in the three prostate cancer lines compared to malignantprostatic tissue from patient #1215 (FIG. 1B). In addition, expressionlevels in all four samples were close to the no template negativecontrols which confirmed little to no endogenous DEFB1 expression (datanot shown). QRT-PCR was also performed on the prostate cancer cell linestransfected with the DEFB1 expression system. Following a 24 hourinduction period, relative expression levels were 0.01360 in DU145,0.01503 in PC3 and 0.138 in LNCaP. Amplification products were verifiedby gel electrophoresis.

QRT-PCR was performed on LCM tissues regions containing benign, PIN andcancer. DEFB1 relative expression was 0.0146 in the benign regioncompared to 0.0009 in the malignant region (FIG. 1C). This represents a94% decrease which again demonstrates a significant down-regulation ofexpression. Furthermore, analysis of PIN revealed that DEFB1 expressionlevel was 0.044 which was a 70% decrease. Comparing expression inpatient #1457 to the average expression level found in benign regions ofsix other patients (FIG. 1A.) revealed a ratio of 1.997 representingalmost twice as much expression (FIG. 1D). However, the expression ratiowas 0.0595 in PIN and was 0.125 in malignant tissue compared to averageexpression levels in benign tissue.

DEFB1 Causes Cell Membrane Permeability and Ruffling: Induction of DEFB1in the prostate cancer cell lines resulted in a significant reduction incell number in DU145 and PC3, but had no effect on cell proliferation inLNCaP (FIG. 2). As a negative control, cell proliferation was monitoredin all three lines containing empty plasmid. There were no observablechanges in cell morphology in DU145, PC3 or LNCaP cells following theaddition of PonA. In addition, DEFB1 induction resulted in morphologicalchanges in both DU145 and PC3. Here cells appeared more rounded andexhibited membrane ruffling indicative of cell death. Apoptotic bodieswere also present in both lines.

Expression of DEFB1 Results in Decreased Cell Viability: The MTT assayshowed a reduction in cell viability by DEFB1 in PC3 and DU145 cells,but no significant effect on LNCaP cells (FIG. 3). After 24 hours,relative cell viability was 72% in DU145 and 56% in PC3. Analysis 48hours after induction revealed 49% cell viability in DU145 and 37% cellviability in PC3. After 72 hours of DEFB1 expression resulted in 44% and29% relative cell viability in DU145 and PC3 cells, respectively.

DEFB1 Causes Rapid Caspase-mediated Apoptosis in Late-stage ProstateCancer Cells: In order to determine whether the effects of DEFB1 on PC3and DU145 were cytostatic or cytotoxic, FACS analysis was performed.Under normal growth conditions, more than 90% of PC3 and DU145 cultureswere viable and non-apoptotic (lower left quadrant) and did not stainwith annexin V or PI (FIG. 4). After inducing DEFB1 expression in PC3cells, the number of apoptotic cells (lower and upper right quadrants)totaled 10% at 12 hours, 20% at 24 hours, and 44% at 48 hours. For DU145cells, the number of apoptotic cells totaled 12% after 12 hours, 34% at24 hours, and 59% after 48 hours of induction. There was no increase inapoptosis observed in cells containing empty plasmid following inductionwith PonA (data not shown).

Caspase activity was determined by confocal laser microscopic analysis(FIG. 5). DU145 and PC3 cell were induced for DEFB1 expression andactivity was monitored based on the binding of green fluorescingFAM-VAD-FMK to caspases in cells actively undergoing apoptosis. Analysisof cells under DIC showed the presence of viable control DU145 (A), PC3(E) and LNCaP (I) cells at 0 hours. Excitation by the confocal laser at488 nm produced no detectable green staining which indicates no caspaseactivity in DU145 (B), PC3 (F) or LNCaP (J). Following induction for 24hours, DU145 (C), PC3 (G) and LNCaP (K) cells were again visible underDIC. Confocal analysis under fluorescence revealed green staining inDU145 (D) and PC3 (H) cell indicating caspase activity. However, therewas no green staining in LNCaP (L), indicating no induction of apoptosisby DEFB1.

In conclusion, this study provides the functional role of DEFB1 inprostate cancer. Furthermore, these findings show that DEFB1 is part ofan innate immune system involved in tumor immunity. Data presented heredemonstrate that DEFB1 expressed at physiological levels is cytotoxic toAR− hormone refractory prostate cancer cells, but not to AR+ hormonesensitive prostate cancer cell nor to normal prostate epithelial cells.Given that DEFB1 is constitutively expressed in normal prostate cellswithout cytotoxicity, it may be that late-stage AR− prostate cancercells possess distinct phenotypic characteristics that render themsensitive to DEFB1 cytotoxicity. Thus, DEFB1 is a viable therapeuticagent for the treatment of late-stage prostate cancer, and potentiallyother cancers as well.

Example 2 siRNA Mediated Knockdown of PAX2 Expression Results inProstate Cancer Cell Death Independent of P53 Status

This example examines the effects of inhibiting PAX2 expression by RNAinterference in prostate cancer cells which differ in p53 gene status.The results demonstrate that the inhibition of PAX2 results in celldeath irrespective of p53 status, indicating that there are additionaltumor suppressor genes or cell death pathways inhibited by PAX2 inprostate cancer.

Materials and Methods

siRNA Silencing of PAX2: In order to achieve efficient gene silencing, apool of four complementary short interfering ribonucleotides (siRNAs)targeting human PAX2 mRNA (Accession No. NM_(—)003989.1), weresynthesized (Dharmacon Research, Lafayette, Colo., USA). A second poolof four siRNAs were used as an internal control to test for thespecificity of PAX2 siRNAs. Two of the sequences synthesized target theGL2 luciferase mRNA (Accession No. X65324), and two werenon-sequence-specific (Table 3). For annealing of siRNAs, 35 M of singlestrands were incubated in annealing buffer (100 mM potassium acetate, 30mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate) for 1 min at 90° C.followed by 1 hour incubation at 37° C.

TABLE 3 PAX2 siRNA Sequences. A pool of four siRNAwas utilized to inhibit PAX2 protein expression. Sense (5′-3′)Sequence A 5′-GAAGUCAAGUCGAGUCUAUUU-3′ SEQ ID NO: 38 Sequence B5′-GAGGAAACGUGAUGAAGAUUU-3′ SEQ ID NO: 39 Sequence C5′-GGACAAGAUUGCUGAAUACUU-3′ SEQ ID NO: 40 Sequence D5′-CAUCAGAGCACAUCAAAUCUU-3′ SEQ ID NO: 41 Antisense (5′-3′) Sequence A5′-AUAGACUCGACUUGACUUCUU-3′ SEQ ID NO: 3 Sequence B5′-AUCUUCAUCACGUUUCCUCUU-3′ SEQ ID NO: 4 Sequence C5′-GUAUUCAGCAAUCUUGUCCUU-3′ SEQ ID NO: 5 Sequence D5′-GAUUUGAUGUGCUCUGAUGUU-3′ SEQ ID NO: 6

Western Analysis: Briefly, cells were harvested by trypsinization andwashed twice with PBS. Lysis buffer was prepared according to themanufacturer's instructions (Sigma), and was then added to the cells.Following a 15 minute incubation period at 4° C. on an orbital shaker,cell lysate were then collected and centrifuged for 10 minutes at12000×g to pellet cellular debris. The protein-containing supernatantwere then collected and quantitated. Next, 25 μg protein extract wasloaded onto an 8-16% gradient SDS-PAGE (Novex). Followingelectrophoresis, proteins were transferred to PVDF membranes, and thenblocked with 5% nonfat dry milk in TTBS (0.05% Tween 20 and 100 mMTris-C1) for 1 hour. Blots were then probed with rabbit anti-PAX2primary antibody (Zymed, San Francisco, Calif.) at a 1:2000 dilution.After washing, the membranes were incubated with anti-rabbit antibodyconjugated to horseradish peroxidase (HRP) (dilution 1:5000; Sigma), andsignal detection was visualized using chemilluminescence reagents(Pierce) on an Alpha Innotech Fluorchem 8900. As a control, blots werestripped and reprobed with mouse anti-β-actin primary antibody (1:5000;Sigma-Aldrich) and HRP-conjugated anti-mouse secondary antibody (1:5000;Sigma-Aldrich) and signal detection was again visualized.

Phase Contrast Microscopy: The effect of PAX2 knock-down on cell growthwas analyzed by phase contrast microscopy. Here, 1-2×10⁴ cells wereseeded onto 6 well culture plates (BD Falcon, USA). The following daycells were treated with media only, negative control non-specific siRNAor PAX2 siRNA and allowed to incubate for six days. The cells were thenviewed under an inverted Zeiss IM 35 microscope (Carl Zeiss, Germany) at32× magnification. Phase contrast pictures of a field of cells wereobtained using the SPOT Insight Mosaic 4.2 camera (DiagnosticInstruments, USA).

MTT Cytotoxicity Assay: DU145, PC3 and LNCaP cells (1×105) weretransfected with 0.5 μg of the PAX2 siRNA pool or control siRNA poolusing Codebreaker transfection reagent according to the manufacturer'sprotocol (Promega). Next, cell suspensions were diluted and seeded ontoa 96-well plate at 1-5×10³ cells per well and allowed to grow for 2-, 4-or 6 days. After culture, cell viability was determined by measuring theconversion of 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazoliumbromide, MTT (Promega), to a colored formazan product. Absorbance wasread at 540 nm on a scanning multiwell spectrophotometer.

Pan-Caspase detection in the prostate cancer cell lines and quantitativereal-time PCR were performed as described in Example 1. The primer pairsfor BAX, BID and BAD were generated from the published sequences (Table4). Reactions were performed in MicroAmp Optical 96-well Reaction Plate(PE Biosystems). Forty cycles of PCR were performed under standardconditions using an annealing temperature of 60° C. Quantification wasdetermined by the cycle number where exponential amplification began(threshold value) and averaged from the values obtained from thetriplicate repeats. There was an inverse relationship between messagelevel and threshold value. In addition, GAPDH was used as a housekeepinggene to normalize the initial content of total cDNA. Gene expression wascalculated as the relative expression ratio between the pro-apoptoticgenes and GAPDH. All reactions were carried out in triplicate.

TABLE 4 Quantitative RT-PCR Primers. Nucleotidesequences of primers used to amplify PAX2 and GAPDH. Sense (5′-3′) GAPDH5′-CCACCCATGGCAAATTCCATGGCA-3′ SEQ ID NO: 42 BAD5′-CTCAGGCCTATGCAAAAAGAGGA-3′ SEQ ID NO: 43 BID5′-AACCTACGCACCTACGTGAGGAG-3′ SEQ ID NO: 44 BAX5′-GACACCTGAGCTGACCTTGG-3′ SEQ ID NO: 45 Antisense (5′-3′) GAPDH5′-TCTAGACGGCAGGTCAGGTCAACC-3′ SEQ ID NO: 46 BAD5′-GCCCTCCCTCCAAAGGAGAC-3′ SEQ ID NO: 47 BID5′-CGTTCAGTCCATCCCATTTCTG-3′ SEQ ID NO: 48 BAX5′-GAGGAAGTCCAGTGTCCAGC-3′ SEQ ID NO: 49

siRNA Inhibition of PAX2 Protein: In order to confirm that the siRNAeffective targeted the PAX2 mRNA, Western Analysis was performed tomonitor PAX2 protein expression levels over a six day treatment period.Cells were given a single round of transfection with the pool of PAX2siRNA. The results confirmed specific targeting of PAX2 mRNA by showingknock-down of PAX2 protein by day four in DU145 (FIG. 6 a) and by daysix in PC3 (FIG. 6 b).

Knock-down of PAX2 inhibit Prostate Cancer Cell Growth: Cells wereanalyzed following a six day treatment period with media only, negativecontrol non-specific siRNA or PAX2 siRNA (FIG. 7). DU145 (a), PC3 (d)and LNCaP (g) cells all reached at least 90% confluency in the culturedishes containing media only. Treatment of DU145 (b), PC3 (e) and LNCaP(h) with negative control non-specific siRNA had no effect on cellgrowth, and cells again reached confluency after six days. However,treatment with PAX2 siRNA resulted in a significant decrease in cellnumber. DU145 cells were approximately 15% confluent (c) and PC3 cellswere only 10% confluent (f). LNCaP cell were 5% confluent followingsiRNA treatment.

Cytotoxicity Assays: Cell viability was measured after two-, four-, andsix-day exposure times, and is expressed as a ratio of the 570-630 nmabsorbance of treated cells divided by that of the untreated controlcells (FIG. 8). Relative cell viability following 2 days of treatmentwas 77% in LNCaP, 82% in DU145 and 78% in PC3. After four days, relativecell viability was 46% in LNCaP, 53% in DU145 and 63% in PC3. After sixdays of treatment, relative cell viability decreased to 31% in LNCaP,37% in PC3, and was 53% in DU145. As negative controls, cell viabilitywas measured in after a six day treatment period with negative controlnon-specific siRNA or transfection reagent alone. For both conditions,there was no statistically significant change in cell viability comparedto normal growth media.

Pan-Caspase Detection: Caspase activity was detected by confocal lasermicroscopic analysis. DU145, PC3 and LNCaP cells were treated with PAX2siRNA and activity was monitored based on the binding of FAM-labeledpeptide to caspases in cells actively undergoing apoptosis which will befluoresce green. Analysis of cells with media only under DIC shows thepresence of viable DU145 (A), PC3 (E) and LNCaP (I) cells at 0 hours(FIG. 9). Excitation by the confocal laser at 488 nm produced nodetectable green staining which indicates no caspase activity inuntreated DU145 (B), PC3 (F) or LNCaP (J). Following four days oftreatment with PAX2 siRNA, DU145 (C), PC3 (G) and LNCaP (K) cells wereagain visible under DIC. Under fluorescence, the treated DU145 (D), PC3(H) and LNCaP (L) cells presented green staining indicating caspaseactivity.

Effect of PAX2 Inhibition on Pro-apoptotic Factors: DU145, PC3 and LNCaPcells were treated with siRNA against PAX2 for six days and expressionof pro-apoptotic genes dependent and independent of p53 transcriptionregulation were measured to monitor cell death pathways. For BAX, therewas a 1.81-fold increase in LNCaP, a 2.73-fold increase in DU145, and a1.87-fold increase in PC3 (FIG. 10 a). Expression levels of BIDincreased by 1.38-fold in LNCaP and 1.77-fold in DU145 (FIG. 10 b).However, BID expression levels decreased by 1.44-fold in PC3 followingtreatment (FIG. 10 c). Analysis of BAD revealed a 2.0-fold increase inexpression in LNCaP, a 1.38-fold increase in DU145, and a 1.58-foldincrease in PC3.

These results demonstrate dependency of prostate cancer cell survival onPAX2 expression. Following p53 activation as a result of PAX2 knock-downin the p53-expressing cell line LNCaP, the p53-mutated line DU145, andthe p53-null line PC3, caspase activity was detected in all three lines,indicating of the initiation of programmed cell death. BAX expressionwas upregulated in all three cell lines independent of p53 status. Theexpression of pro-apoptotic factor BAD was also increased in all threelines following PAX2 inhibition. Following treatment with PAX2 siRNA,BID expression was increased in LNCaP and DU145, but actually decreasedin PC3. These results indicate that cell death observed in prostatecancer is influenced by but is not dependent on p53 expression. Theinitiation of apoptosis in prostate cancer cells through different celldeath pathways irrespective of p53 status indicates that PAX2 inhibitsother tumor suppressors.

Besides SEQ ID NOS: 3-6, other examples of anti-PAX2 siRNAs include, butare not limited to, siRNAs having the sequences of (5′ to 3′ direction):

(SEQ ID NO: 7) ACCCGACTATGTTCGCCTGG, (SEQ ID NO: 8)AAGCTCTGGATCGAGTCTTTG, (SEQ ID NO: 9) ATGTGTCAGGCACACAGACG,(SEQ ID NO: 10) GUCGAGUCUAUCUGCAUCCUU, (SEQ ID NO: 11)GGAUGCAGAUAGACUCGACUU,

PAX proteins are a family of transcription factors conserved duringevolution and able to bind specific DNA sequences through a domainscalled a “paired domain (PD)” and a “homeodomain (HD).” The PD is aconsensus sequence shared by certain PAX proteins (e.g., PAX2 and PAX6).The PD directs DNA binding of amino acids located in the α3-helixforming a DNA-Protein complex. For PAX2, the amino acids in the HDrecognize and interact specifically with a CCTTG (SEQ ID NO:1) DNA coresequence. Oligonucleotides including this sequence or its complement areexpected to be inhibitors of PAX 2 proteins.

In one embodiment, the oligonucleotide has the sequence of V-CCTTG-W(SEQ ID NO: 12), wherein V is from 1 to 35 contiguous flankingnucleotides of DEFB1 and W is from 1 to 35 nucleotides. The nucleotidescan be contiguous nucleotides that normally flank the PAX2 DNA bindingsite of DEFB1. Alternatively, they can be unrelated to DEFB1, andselected routinely to avoid interference with the recognition sequence

Other examples of oligonucleotides that inhibit PAX2 binding to theDEFB1 promoter include, but are not limited to, oligonucleotide havingthe sequences of (5′ to 3′ direction):

(SEQ ID NO: 13) CTCCCTTCAGTTCCGTCGAC (SEQ ID NO: 14)CTCCCTTCACCTTGGTCGAC (SEQ ID NO: 15)ACTGTGGCACCTCCCTTCAGTTCCGTCGACGAGGTTGTGC (SEQ ID NO: 16)ACTGTGGCACCTCCCTTCACCTTGGTCGACGAGGTTGTGC

Example 3 Inhibition of PAX2 Oncogene Results in DEFB1-Mediated Death ofProstate Cancer Cells

The identification of tumor-specific molecules that serve as targets forthe development of new cancer drugs is considered to be a major goal incancer research. Example 1 demonstrated that there is a high frequencyof DEFB1 expression loss in prostate cancer, and that induction of DEFB1expression results in rapid apoptosis in androgen receptornegative-stage prostate cancer. These data show that DEFB1 plays a rolein prostate tumor suppression. In addition, given that it is a naturallyoccurring component of the immune system of normal prostate epithelium,DEFB1 is expected to be a viable therapeutic agent with little to noside effects. Example 2 demonstrated that inhibition of PAX2 expressionresults in prostate cancer cell death independent of p53. These dataindicate that there is an addition pro-apoptotic factor or tumorsuppressor that is inhibited by PAX2. In addition, the data show thatthe oncogenic factor PAX2, which is over-expressed in prostate cancer,is a transcriptional repressor of DEFB1. The purpose of this study is todetermine if DEFB1 loss of expression is due to aberrant expression ofthe PAX2 oncogene, and whether inhibiting PAX2 results in DEFB1-mediatedcell death.

Materials and Methods

RNA Isolation and Quantitative RT-PCR were performed as described inExample 1. The primer pair for DEFB1 was generated from the publishedDEFB1 sequence (Accession No. U50930). Forty cycles of PCR wereperformed under standard conditions using an annealing temperature of56° C. In addition, GAPDH was amplified as a housekeeping gene tonormalize the initial content of total cDNA. DEFB1 expression wascalculated as the relative expression ratio between DEFB1 and GAPDH andwas compared in cells lines before and after siRNA knock-down of PAX2expression. All reactions were run three times in triplicate.

Generation of the DEFB1 Reporter Construct: The pGL3 luciferase reporterplasmid was used to monitor DEFB1 reporter activity. Here, a region 160bases upstream of the DEFB1 transcription initiation site and includedthe DEFB1 TATA box. The region also included the GTTCC (SEQ ID NO: 2)sequence which is necessary for PAX2 binding. The PCR primers weredesigned to contain Kpn1 and Nhe1 restriction sites. The DEFB1 promoterPCR products were restriction digested Kpn I and NheI and ligated into asimilary restriction digested pGL3 plasmid (FIG. 2). The constructs weretransfected into E. coli and individual clones were selected andexpanded. Plasmids were isolated and sequence integrity of theDEFB1/pGL3 construct was verified by automated sequencing.

Luciferase Reporter Assay: Here, 11 g of the DEFB1 reporter construct orthe control pGL3 plasmid was transfected into 1×10⁶ DU145 cells. Next,0.5×10³ cells were seeded onto each well of a 96-well plate and allowedto grow overnight. Then fresh medium was added containing PAX2 siRNA ormedia only and the cells were incubated for 48 hours. Luciferase wasdetected by the BrightGlo kit according to the manufacturer's protocol(Promega) and the plates were read on a Veritas automated 96-wellluminometer. Promoter activity was expressed as relative luminescence.

Analysis of Membrane Permeability: Acridine orange (AO)/ethidium bromide(EtBr) dual staining was performed to identify changes in cell membraneintegrity, as well as apoptotic cells by staining the condensedchromatin. AO stains viable cells as well as early apoptotic cells,whereas EtBr stains late stage apoptotic cells that have lost membranepermeability. Briefly, cells were seeded into 2 chamber culture slides(BD Falcon, USA). Cells transfected with empty pIND plasmid/pvgRXR orpIND DEFB1/pvgRXR were induced for 24 or 48 hour with media containing10 μM Ponasterone A. Control cells were provided fresh media at 24 and48 h. In order to determine the effect of PAX2 inhibition on membraneintegrity, separate culture slides containing DU145, PC3 and LNCaP weretreated with PAX2 siRNA and incubated for 4 days. Following this, cellswere washed once with PBS and stained with 2 ml of a mixture (1:1) of AO(Sigma, USA) and EtBr (Promega, USA) (5 ug/ml) solution for 5 min.Following staining, the cells were again washed with PBS. Fluorescencewas viewed by a Zeiss LSM 5 Pascal Vario 2 Laser Scanning ConfocalMicroscope (Carl Zeiss Jena, Germany). The excitation color wheelcontain BS505-530 (green) and LP560 (red) filter blocks which allowedfor the separation of emitted green light from AO into the green channeland red light from EtBr into the red channel. The laser power output andgain control settings within each individual experiment were identicalbetween control and DEFB1 induced cells. The excitation was provided bya Kr/Ar mixed gas laser at wavelengths of 543 nm for AO and 488 nm forEtBr. Slides were analyzed under 40× magnification and digital imageswere stored as uncompressed TIFF files and exported into Photoshop CSsoftware (Adobe Systems, San Jose, Calif.) for image processing and hardcopy presentation.

ChIP Analysis of PAX2: Chromatin immunoprecipitation (ChIP) allows theidentification of binding sites for DNA-binding proteins based upon invivo occupancy of a promoter by a transcription factor and enrichment oftranscription factor bound chromatin by immunoprecipitation. Amodification of the protocol described by the Farnham laboratory wasused; also on line at http://mcardle.oncology.wisc.edu/farnham/). TheDU145 and PC3 cell lines over-expresses the PAX2 protein, but does notexpress DEFB1. Cells were incubated with PBS containing 1.0%formaldehyde for 10 minutes to crosslink proteins to DNA. Samples werethen sonicated to yield DNA with an average length of 600 bp. Sonicatedchromatin precleared with Protein A Dynabeads was incubated withPAX2-specific antibody or “no antibody” control [isotype-matched controlantibodies]. Washed immunoprecipitates were then collected. Afterreversal of the crosslinks, DNA was analyzed by PCR usingpromoter-specific primers to determine whether DEFB1 is represented inthe PAX2-immunoprecipitated samples. Primers were designed to amplifythe 160 bp region immediately upstream of the DEFB1 mRNA start sitewhich contained the DEFB1 TATA box and the functional GTTCC (SEQ ID NO:2) PAX2 recognition site. For these studies, positive controls includedPCR of an aliquot of the input chromatin (prior to immunoprecipitation,but crosslinks reversed). All steps were performed in the presence ofprotease inhibitors.

siRNA Inhibition of PAX2 Increases DEFB1 Expression: QRT-PCR analysis ofDEFB1 expression before siRNA treatment revealed relative expressionlevels of 0.00097 in DU145, 0.00001 in PC3, and 0.00004 LNCaP (FIG. 13).Following siRNA knock-down of PAX2, relative expression was 0.03294(338-fold increase) in DU145, 0.00020 (22.2-fold increase) in PC3 and0.00019 (4.92-fold increase) in LNCaP. As a negative control, the humanprostate epithelial cell line (hPrEC) which is PAX2 null, revealedexpression levels at 0.00687 before treatment and 0.00661 followingsiRNA treatment confirming no statistical change in DEFB1 expression.

DEFB1 Causes Cell Membrane Permeability: Membrane integrity wasmonitored by confocal analysis (FIG. 14). Here, intact cells stain greendue to AO which is membrane permeable. In addition, cells withcompromised plasma membranes would stain red by EtBr which is membraneimpermeable. Here, uninduced DU145 (A) and PC3 (D) cells stainedpositively with AO and emitted green color, but did not stain with EtBr.However, DEFB1 induction in both DU145 (B) and PC3 (E) resulted in theaccumulation of EtBr in the cytoplasm at 24 hours indicated by the redstaining. By 48 hours, DU145 (C) and PC3 (F) possessed condensed nucleiand appeared yellow, which was due to the presence of both green and redstaining resulting from the accumulation of AO and EtBr, respectively.

Inhibition of PAX2 Results in Membrane Permeability: Cells were treatedwith PAX2 siRNA for 4 days and membrane integrity was monitored again byconfocal analysis. Here, both DU145 and PC3 possessed condensed nucleiand appeared yellow. However, LNCaP cells' cytoplasm and nuclei remainedgreen following siRNA treatment. Also red staining at the cell peripheryindicates the maintenance of cell membrane integrity. These findingsindicate that the inhibition of PAX2 results in specificallyDEFB1-mediated cell death in DU1145 and PC3, but not LNCaP cells. Deathobserved in LNCaP is due to the transactivation of the existingwild-type p53 in LNCaP following PAX2 inhibition.

siRNA Inhibition of PAX2 Increases DEFB1 Promoter Activity: Analysis ofDEFB1 promoter activity in DU145 cells containing the DEFB1/pGL3construct revealed a 2.65 fold increase in relative light unitsfollowing 48 hours of treatment compared to untreated cells. In PC3cells, there was a 3.78-fold increase in relative light units comparedto untreated cells.

PAX2 Binds to the DEFB1 Promoter: ChIP analysis was performed on DU145and PC3 cells to determine if the PAX2 transcriptional repressor isbound to the DEFB1 promoter (FIG. 15). Lane 1 contains a 100 bpmolecular weight marker. Lane 2 is a positive control representing 160bp region of the DEFB1 promoter amplified from DU145 beforecross-linking and immunoprecipitation. Lane 3 is a negative controlrepresenting PCR performed without DNA. Lanes 4 and 5 are negativecontrols representing PCR from immunoprecipitations performed with IgGfrom cross-linked DU145 and PC3, respectively. PCR amplification of 25pg of DNA (lane 6 and 8) and 50 pg of DNA (lane 7 and 9)immunoprecitipated with anti-PAX2 antibody after crosslinking show 160bp promoter fragment in DU145 and PC3, respectively.

These results demonstrate that the oncogenic factor PAX2 suppressesDEFB1 expression. The suppression occurs at the transcriptional level.Furthermore, computational analysis of the DEFB1 promoter revealed thepresence of a GTTCC (SEQ ID NO: 2) DNA binding site for the PAX2transcriptional repressor next to the DEFB1 TATA box (FIG. 1). One ofthe hallmarks of defensin cytotoxicity is the disruption of membraneintegrity. These results show that ectopic expression of DEFB1 inprostate cancer cells results in a loss of membrane potential due tocompromised cell membranes. The same phenomenon is observed afterinhibiting PAX2 protein expression. Therefore, suppression of PAX2expression or function, results in the re-establishment of DEFB1expression and subsequently DEFB1-mediated cell death. Also, the presentresults establish the utility of DEFB1 as a directed therapy forprostate cancer treatment, and potentially other cancer treatments,through innate immunity.

Example 4 Effect of DEFB1 Expression in Implanted Tumor Cells

The anti-tumoral ability of DEFB1 is evaluated by injecting tumor cellsthat overexpress DEFB1 into nude mice. DEFB1 is cloned into pBI-EGFPvector, which has a bidirectional tetracycline responsible promoter.Tet-Off Cell lines are generated by transfecting pTet-Off into DU145,PC3 and LNCaP cells and selecting with G418. The pBI-EGFP-DEFB1 plasmidis co-transfected with pTK-Hyg into the Tet-off cell lines and selectedwith hygromycin. Only single-cell suspensions with a viability of >90%are used. Each animal receives approximately 500,000 cells administeredsubcutaneously into the right flank of female nude mice. There are twogroups, a control group injected with vector only clones and a groupinjected with the DEFB1 over-expressing clones. 35 mice are in eachgroup as determined by a statistician. Animals are weighed twice weekly,tumor growth monitored by calipers and tumor volumes determined usingthe following formula: volume=0.5×(width)2×length. All animals aresacrificed by CO2 overdose when tumor size reaches 2 mm3 or 6 monthsfollowing implantation; tumors are excised, weighed and stored inneutral buffered formalin for pathological examination. Differences intumor growth between the groups are descriptively characterized throughsummary statistics and graphical displays. Statistical significance isevaluated with either the t-test or non-parametric equivalent.

Example 5 Effect of PAX2 siRNA on Implanted Tumor Cells

Hairpin PAX2 siRNA template oligonucleotides utilized in the in vitrostudies are utilized to examine the effect of the up-regulation of DEFB1expression in vivo. The sense and antisense strand (see Table 4) areannealed and cloned into pSilencer 2.1 U6 hygro siRNA expression vector(Ambion) under the control of the human U6 RNA pol III promoter. Thecloned plasmid is sequenced, verified and transfected into PC3, Du145,and LNCap cell lines. Scrambled shRNA is cloned and used as a negativecontrol in this study. Hygromycin resistant colonies are selected, cellsare introduced into the mice subcutaneously and tumor growth ismonitored as described above.

Example 6 Effect of Small Molecule Inhibitors of PAX2 Binding onImplanted Tumor Cells

The DNA recognition sequence for PAX2 binding resides in the DEFB1promoter between nucleotides −75 and −71 [+1 refers to thetranscriptional start site]. Short oligonucleotides complementary to thePAX2 DNA-binding domain are provided. Examples of such oligonucleotidesinclude the 20-mer and 40-mer oligonucleotides containing the GTTCC (SEQID NO: 2) recognition sequence provided below. These lengths wererandomly selected, and other lengths are expected to be effective asblockers of binding. As a negative control, oligonucleotides with ascrambled sequence (CTCTG)—(SEQ ID NO: 17) were designed to verifyspecificity. The oligonucleotides are transfected into the prostatecancer cells and the HPrEC cells with lipofectamine reagent orCodebreaker transfection reagent (Promega, Inc). In order to confirmDNA-protein interactions, double stranded oligonucleotides will belabeled with [³²P] dCTP and electrophoretic mobility shift assays areperformed. In addition, DEFB1 expression is monitored by QRT-PCR andWestern analysis following treatment with oligonucleotides. Finally,cell death is detected by MTT assay and flow cytometry as previouslydescribed.

Recognition Sequence #1: (SEQ ID NO: 13) CTCCCTTCAGTTCCGTCGACRecognition Sequence #2: (SEQ ID NO: 14) CTCCCTTCACCTTGGTCGACScramble Sequence #1: (SEQ ID NO: 18) CTCCCTTCACTCTGGTCGACRecognition Sequence #3: (SEQ ID NO: 15)ACTGTGGCACCTCCCTTCAGTTCCGTCGACGAGGTTGTGC Recognition Sequence #4:(SEQ ID NO: 16) ACTGTGGCACCTCCCTTCACCTTGGTCGACGAGGTTGTGCScramble Sequence #2: (SEQ ID NO: 19)ACTGTGGCACCTCCCTTCACTCTGGTCGACGAGGTTGTGC

Further examples of oligonucleotides of the invention include:

Recognition Sequence #1: (SEQ ID NO: 20) 5′-AGAAGTTCACCCTTGACTGT-3′Recognition Sequence #2: (SEQ ID NO: 21) 5′-AGAAGTTCACGTTCCACTGT-3′Scramble Sequence #1: (SEQ ID NO: 22) 5′-AGAAGTTCACGCTCTACTGT-3′Recognition Sequence #3: (SEQ ID NO: 23)5′-TTAGCGATTAGAAGTTCACCCTTGACTGTGGCACCTCCC-3′ Recognition Sequence #4:(SEQ ID NO: 24) 5′-GTTAGCGATTAGAAGTTCACGTTCCACTGTGGCACCTCCC-3′Scramble Sequence #2: (SEQ ID NO: 25)5′-GTTAGCGATTAGAAGTTCACGCTCTACTGTGGCACCTCCC-3′

This set of alternative inhibitory oligonucleotides represents therecognition sequence (along with the CCTTG (SEQ ID NO: 1) core sequence)for the PAX2 binding domain and homeobox. These include actual sequencesfrom the DEFB1 promoter.

The PAX2 gene is required for the growth and survival of various cancercells including prostate. In addition, the inhibition of PAX2 expressionresults in cell death mediated by the innate immunity component DEFB1.Suppression of DEFB1 expression and activity is accomplished by bindingof the PAX2 protein to a GTTCC (SEQ ID NO: 2) recognition site in theDEFB1 promoter. Therefore, this pathway provides a viable therapeutictarget for the treatment of prostate cancer. In this method, thesequences bind to the PAX2 DNA binding site and block PAX2 binding tothe DEFB1 promoter thus allowing DEFB1 expression and activity. Theoligonucleotide sequences and experiment described above are examples ofand demonstrate a model for the design of additional PAX2 inhibitordrugs.

Given that the GTTCC (SEQ ID NO: 2) sequence exists in interleukin-3,interleukin-4, the insulin receptor and others, PAX2 regulates theirexpression and activity as well. Therefore the PAX2 inhibitors disclosedherein have utility in a number of other diseases including thosedirected related to inflammation including prostatitis and benignprostatic hypertrophy (BPH).

Example 7 Loss of DEFB1 Expression Results in Increased Tumorigenesis

Generation of Loss of Function Mice: The Cre/loxP system has been usefulin elucidating the molecular mechanisms underlying prostatecarcinogenesis. Here a DEFB1 Cre conditional KO is used for inducibledisruption within the prostate. The DEFB1 Cre conditional KO involvesthe generation of a targeting vector containing loxP sites flankingDEFB1 coding exons, targeted ES cells with this vector and thegeneration of germline chimeric mice from these targeted ES cells.Heterozygotes are mated to prostate-specific Cre transgenics andheterozygous intercross is used to generate prostate-specific DEFB1 KOmice. Four genotoxic chemical compounds have been found to induceprostate carcinomas in rodents: N-methyl-N-nitrosourea (MNU),N-nitrosobis 2-oxopropyl. amine (BOP), 3,2X-dimethyl-4-amino-biphenyl(MAB) and 2-amino-1-methyl-6-phenylimidazow 4,5-bxpyridine (PhIP).DEFB1-transgenic mice are treated with these carcinogenic compounds viaintra-gastric administration or i.v. injection for prostate adenoma andadenocarcinoma induction studies. Prostate samples are studied fordifferences in tumor growth and changes gene expression thoughhistological, immunohistological, mRNA and protein analyses.

Generation of GOF mice: For PAX2 inducible GOF mice, PAX2 GOF(bi-transgenic) and wild-type (mono-transgenic) littermates areadministered doxycycline (Dox) from 5 weeks of age to induceprostate-specific PAX2 expression. Briefly, PROBASIN-rtTAmono-transgenic mice (prostate cell-specific expression of tet-dependentrtTA inducer) are crossed to our PAX2 transgenic responder lines. Forinduction, bi-transgenic mice are fed Dox via the drinking water (500mg/L freshly prepared twice a week). Initial experiments verify lowbackground levels, good inducibility and cell-type specific expressionof PAX2 and the EGFP reporter using transgenic founder line inbi-transgenic mice. Regarding experimental group sizes, 5-7 age- andsex-matched individuals in each group (wild-type and GOF) allow forstatistical significance. For all animals in this study, prostatetissues are collected initially at weekly intervals for analysis andcomparison, to determine carcinogenic time parameters.

PCR Genotyping, RT-PCR and qPCR: PROBASIN-rtTA transgenic mice aregenotyped using the following PCR primers and conditions:

PROBASIN5 (forward) (SEQ ID NO: 26) 5′-ACTGCCCATTGCCCAAACAC-3′;RTTA3 (reverse) (SEQ ID NO: 27) 5′-AAAATCTTGCCAGCTTTCCCC-3′;

95° C. denaturation for 5 min, followed by 30 cycles of 95° C. for 30see, 57° C. for 30 sec, 72° C. for 30 sec, followed by a 5 min extensionat 72° C., yielding a 600 bp product.

PAX2 inducible transgenic mice are genotyped using the following PCRprimers and conditions:

PAX2For (SEQ ID NO: 28) 5′-GTCGGTTACGGAGCGGACCGGAG-3′; Rev5′IRES(SEQ ID NO: 29) 5′-TAACATATAGACAAACGCACACCG-3′;

95° C. denaturation for 5 min, followed by 34 cycles of 95° C. for 30sec, 63° C. for 30 sec, 72° C. for 30 sec, followed by a 5 min extensionat 72° C., yielding a 460 bp product.

Immortomouse hemizygotes are be genotyped using the following PCRprimers and conditions:

Immol1, (SEQ ID NO: 30) 5′-GCGCTTGTGTC GCCATTGTATTC-3′; Immol2,(SEQ ID NO: 31) 5′-GTCACACCACAGAAGTAAGGTTCC-3′;

94° C. 30 sec, 58° C. 1 min, 72° C. 1 min 30 sec, 30 cycles to yield a˜1 kb transgene band. For genotyping PAX2 knockout mice, the followingPCR primers and conditions are used:

PAX2For (SEQ ID NO: 32) 5′-GTCGGTTACGGAGCGGACCGGAG-3′; PAX2Rev(SEQ ID NO: 33) 5′-CACAGAGCATTGGCGATCTCGATGC-3′;

94° C. 1 min, 65° C. 1 min, 72° C. 30 sec, 36 cycles to yield a 280 bpband.

DEFB1 Peptide Animal Studies:

Six-week-old male athymic (nude) mice purchased from Charles RiverLaboratories are injected sub-cutaneously over the scapula with 10⁶viable PC3 cells. One week after injection, the animals are randomlyallocated to one of three groups-group I: control; group II:intraperitoneal injections of DEFB1,100 μg/day, 5 days a week, for weeks2-14; group III: intraperitoneal injections of DEFB1, 100 mg/day, 5 daysa week, for weeks 8-14. Animals are maintained in sterile housing, fouranimals to a cage, and observed on a daily basis. At 10-day intervals,the tumors are measured by using calipers, and the volumes of the tumorsare calculated by using V=(L×W2)/2.

Example 8 Targeting PAX2 Expression for the Chemoprevention ofIntraepithelial Neoplasia and Cancer

Cancer chemoprevention is defined as the prevention of cancer ortreatment at the pre-cancer state or even earlier. The long period ofprogression to invasive cancer is a major scientific opportunity butalso an economic obstacle to showing the clinical benefit of candidatechemopreventive drugs. Therefore, an important component ofchemopreventive agent development research in recent years has been toidentify earlier (than cancer) end points or biomarkers that accuratelypredict an agent's clinical benefit or cancer incidence-reducing effect.In many cancers, IEN is an early end point such as in prostate cancer.Given that the PAX2/DEFB1 pathway is deregulated during IEN and perhapsat even an earlier histopathological state makes it a powerfulpredictive biomarker and an excellent target for chemoprevention ofcancer. Shown are a number of compounds that suppress PAX2 and increasesDEFB1 expression that may have utility as chemoprevention agents forprostate cancer.

As shown in Table 1, the PAX2 gene is expressed in a number of cancers.In addition, several cancers have been shown to have aberrant PAX2expression (FIG. 18). Chemoprevention via target PAX2 expression mayhave a significant impact on cancer related deaths. Angiotensin II(AngII) is a major regulator of blood pressure and cardiovascularhomeostasis and is recognized as a potent mitogen. AngII mediates itsbiological effects through binding to two subtypes of receptors,Angiotensin Type I receptor (AT1R) and Angiotensin Type II receptor(AT2R) which belong to the super-family of G-protein-coupled receptorsbut have different tissue distribution and intracellular signalingpathways. In addition to its effects on blood pressure, AngII has beenshown to play a role in various pathological situations involving tissueremodeling, such as wound healing, cardiac hypertrophy and development.In fact, recent studies have revealed local expression of severalcomponents of the Renin-Angiotensin System (RAS) in various cancer cellsand tissues including the prostate. Upregulation of AT1R provides aconsiderable advantage to cancer cells that have learn to evadeapoptosis and growth regulatory elements.

Materials and Methods

Reagents and Treatments: Cells were treated with 5 or 10 uM of AngII, 5uM of the ATR1 antagonist Los, 5 uM of the ATR2 antagonist PD123319, 25uM of the MEK inhibitor U0126, 20 uM of the MEK/ERK inhibitor PD98059 or250 μM of the AMP kinase inducer AICAR.

Western Analysis was performed as described in Example 2. Blots werethen probed with primary antibody (anti-PAX2, -phospho-PAX2, -JNK,-phospho-JNK, -ERK1/2, or -phospho-ERK1/2) (Zymed, San Francisco,Calif.) at 1:1000-2000 dilutions. After washing, the membranes wereincubated with anti-rabbit antibody conjugated to horseradish peroxidase(HRP) (dilution 1:5000; Sigma), and signal detection was visualizedusing chemilluminescence reagents (Pierce) on an Alpha InnotechFluorchem 8900. As a control, blots were stripped and re-probed withmouse anti-β-actin primary antibody (1:5000; Sigma-Aldrich) andHRP-conjugated anti-mouse secondary antibody (1:5000; Sigma-Aldrich),and signal detection was again visualized.

QRT-PCR Analysis was performed as described in Example 1. Reactions wereperformed in MicroAmp Optical 96-well Reaction Plate (PE Biosystems).Forty cycles of PCR were performed under standard conditions using anannealing temperature of 60° C. Quantification was determined by thecycle number where exponential amplification began (threshold value) andaveraged from the values obtained from the triplicate repeats. There wasan inverse relationship between message level and threshold value. Inaddition, GAPDH was used as a housekeeping gene to normalize the initialcontent of total cDNA. Relative expression was calculated as the ratiobetween each genes and GAPDH. All reactions were carried out intriplicate.

Thymidine Incorporation:

Proliferation of cells was determined by [³H]thymidine ribotide ([³H]TdR) incorporation into DNA. 0.5×10⁶ cells/well of suspension DU145cells were plated in their appropriate media. Cells were incubated for72 hrs with or without the presence of AngII at the indicatedconcentrations. Cells were exposed to 37 kBq/ml [methyl-³ H]thymidine inthe same medium for 6 hours. The adherent cells were fixed by 5%trichloroacetic acid and lysed in SDS/NaOH lysis buffer overnight.Radioactivity was measured by Beckman LS3801 liquid scintillationcounter (Canada). Suspension cell culture was harvested by cellharvester (Packard instrument Co., Meriden, Conn.), and radioactivitywas measured by 1450 microbeta liquid scintillation counter (PerkinElmerLife Sciences).

To investigate the effect of AngII on PAX2 expression in DU145 prostatecancer cells, PAX2 expression was examined following treatment withAngII over a 30 min to 48 hour period. As shown in FIG. 19, PAX2expression progressively increased over time following AngII treatment.Blocking RAS signaling by treating DU145 with Los significantly reducedPAX2 expression (FIG. 20A). Here, PAX2 expression was 37% after 48 hoursand was 50% after 72 hours of Los treatment compared to untreatedcontrol DU145 cells (FIG. 21). It is known that the AT2R receptor opposethe action of the AT1R. Therefore, the effect of blocking the AT2Rreceptor on PAX2 expression was examined. Treatment of DU145 with theAT2R blocker PD123319 resulted in a 7-fold increase in PAX2 expressionafter 48 hours and an 8-fold increase after 96 hours of treatment (FIG.20B). Collectively, these findings demonstrate that PAX2 expression isregulated by the ATR1 receptor.

It is known that AngII directly affects the proliferation of prostatecancer cells through AT1R-mediated activation of MAPK and STAT3phosphorylation. Treatment of DU145 with AngII resulted in a two- tothree-fold increase in proliferation rate (FIG. 21). However, treatmentwith Los decreased proliferated rates by 50%. In addition, blocking theAT1R receptor by pre-treating with Los for 30 min suppressed the effectof AngII on proliferation.

To further examine the role of the AT1R signaling in the regulation ofPAX2 expression and activation, the effect of blocking variouscomponents of the MAP kinase signaling pathway on PAX2 expression wasexamined. Here, DU145 cells treated with the MEK inhibitor U0126resulted in a significant reduction of PAX2 expression (FIG. 22).Furthermore, treatment with MEK/ERK inhibitor PD98059 also resulted indecreased PAX2. Treatment of DU145 cells with Los had no effect on ERKprotein levels, but reduced the amount of phospho-ERK (FIG. 23A).However, treatment of DU145 with Los resulted in a significant reductionof PAX2 expression. Similar results were observed with U0126 andPD98059. It is also known that PAX2 expression is regulated by STAT3which is a down-stream target of ERK. Treatment of DU145 with Los,U0126, and PD98059 reduced phospho-STAT3 protein levels (FIG. 23C).These results demonstrate that PAX2 is regulated via AT1R in prostatecancer cells.

In addition, the effect of AT1R signaling on PAX2 activation by JNK wasexamined. Treatment of DU145 with Los, U0126, and PD98059 all resultedin a significant decrease or suppression of phospho-PAX2 protein levels(FIG. 24A). However, Los and U0126 did not decrease phospho-JNK proteinlevels (FIG. 24B). Therefore, the decrease in phospho-PAX2 appears to bedue to decreased PAX2 levels, but not decreased phosphorylation.

5-Aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) is widelyused as an AMP-kinase activator, which regulates energy homeostasis andresponse to metabolic stress. Recent reports have indicatedanti-proliferative and pro-apoptotic action of activated AMPK usingpharmacological agents or AMPK overexpression. AMPK activation has beenshown to induce apoptosis in human gastric cancer cells, lung cancercells, prostate cancer, pancreatic cells, and hepatic carcinoma cellsand enhance oxidative stress induced apoptosis in mouse neuroblastomacells, by various mechanisms that include inhibition of fatty acidsynthase pathway and induction of stress kinases and caspase 3. Inaddition, treatment of PC3 prostate cancer cells increased expression ofp21, p27, and p53 proteins and inhibition of PI3K-Akt pathway. All ofthese pathways are directly or indirectly regulated by PAX2. Treatmentof prostate cancer cells with AICAR resulted in the suppression of PAX2expression (FIG. 23B) as well as its activated form phosphor-PAX2 (FIG.24A). In addition, phospho-STAT3 which regulated PAX2 expression wasalso suppressed (FIG. 23C).

Finally, it was hypothesized that aberrant RAS signaling which leads toupregulation and overexpression of PAX2 suppresses the expression of theDEFB1 tumor suppressor gene. To investigate this, the normal prostateepithelial primary culture hPrEC was treated with AngII and examinedboth PAX2 and DEFB1 expression levels. An inverse relationship betweenDEFB1 and PAX2 expression was discovered in normal prostate cells versusprostate cancer cells. Untreated hPrEC exhibited 10% relative PAX2expression compared to expression in PC3 prostate cancer cells.Conversely, untreated PAX2 exhibited only 2% relative DEFB1 expressioncompared to expression in hPrEC. Following 72 hours of treatment with 10uM of AngII, there was a 35% decrease in DEFB1 expression compared tountreated hPrEC, and by 96 hours there was a 50% decrease in DEFB1expression compared to untreated hPrEC cells. However, there was 66%increase in PAX2 expression at 72 hours, and by 96 hours there was a 79%increase in PAX2 expression compared to untreated hPrEC cells.Furthermore, the increase in PAX2 expression in hPrEC after 72 hours was77% of PAX2 levels observed in PC3 prostate cancer cells. After 96 hoursof AngII treatment PAX2 expression was 89% of PAX2 expression in PC3.These results demonstrate that deregulated RAS signaling suppressesDEFB1 expression via the upregulation of PAX2 expression in prostatecells.

Inhibition of apoptosis is a critical pathophysiological factor thatcontributes to the development of cancer. Despite significant advancesin cancer therapeutics, little progress has been made in the treatmentof advanced disease. Given that carcinogenesis is a multiyear,multistep, multipath disease of progression, chemoprevention through theuse of drug or other agents to inhibit, delay, or reverse this processhas been recognized as a very promising area of cancer research.Successful drug treatment for the chemoprevention of prostate cancerrequires the use of therapeutics with specific effects on target cellswhile maintaining minimal clinical effects on the host with the overallgoal of suppressing cancer development. Therefore, understanding themechanisms in early stage carcinogenesis is critical in determining theefficacy of a specific treatment. The significance of aberrant PAX2expression and its abrogation of apoptosis, with subsequent contributionto tumor formation, suggest that it may be a suitable target forprostate cancer treatment. PAX2 was regulated by the AT1R in prostatecancer (FIG. 26). In this, deregulated RAS signaling resulted inincreased PAX2 oncogene expression, and a decrease in the expression ofDEFB1 tumor suppressor. Therefore, the use of AT1R antagonists decreasesPAX2 expression and results in increased prostate cancer cell death viare-expression of DEFB1 (FIG. 27). These results offer a novel findingthat targeting PAX2 expression via the Renin-Angiotensin signalingpathway, the AMP Kinase pathway, or other methods involving theinactivation of the PAX2 protein (i.e. anti-PAX2 antibody vaccination)may be a viable target for cancer prevention (Table 5).

TABLE 5 Compounds Utilized to Inhibit PAX2 Expression forChemoprevention NAME Drug Class Drug 1 Losartan Angiotensin Type 1Receptor blocker Drug 2 PD123319 Angiotensin Type 2 Receptor blockerDrug 3 U0126 MEK inhibitor Drug 4 PD98059 MEK/ERK inhibitor Drug 5 AICARAMP kinase inducer Target Drug Function Drug A Anti-PAX2 Antibody PAX2Vaccine Drug B Angiotensinogen Renin-AngII pathway inhibitor Drug CAngiotensin Converting Renin-AngII pathway inhibitor Enzyme

This study demonstrates that the upregulation of the PAX2 oncogene inprostate cancer is due to deregulated RAS signaling. PAX2 expression isregulated by the ERK 1/2 signaling pathway which is mediated by theAngiotensin type I receptor. In addition, blocking the AT1R withLosartan (Los) suppresses PAX2 expression. In addition, AICAR which isan AMPK activator has also shown promise as a potential PAX2 inhibitor.Collectively, these studies strongly implicate these classes of drugs aspotential suppressors of PAX2 expression and may ultimately serve asnovels chemoprevention agents.

Example 9 PAX2-DEFB1 Expression Level as a Grading Tool for ProstateTissue and Predictor of Prostate Cancer Development Materials andMethods

QRT-PCR Analysis:

Prostate sections were collected from patients that underwent radicalprostatectomies. Following pathological examination, laser capturemicrodisection was performed to isolate areas of Normal, ProliferativeIntraepithelial Neoplasia (PIN) and Cancerous tissue. QRT-PCR wasperformed as previously described to assess expression. DEFB1 and PAX2expression in each region and GAPDH was used as an internal control.

Blood collection and RNA isolation: For QRT-PCR, blood (2.5 ml) fromeach individual was collected into a PAXgene™ Blood RNA tube (QIAGEN)following the manufacturer's protocol. Whole blood was thoroughly mixedwith PAXgene stabilization reagent and stored at room temperature for 6hours prior to RNA extraction. Total RNA was then extracted using thePAXgene™ Blood RNA kit according to the manufacturer's directions(QIAGEN). In order to remove contaminating genomic DNA, total RNAsamples absorbed to the PAXgene™ Blood RNA System spin column wasincubated with DNase I (QIAGEN) at 25° C. for 20 min to remove genomicDNA. Total RNA was eluted, quantitated, and QRT-PCR is performed aspreviously mentioned to compare PAX2 and DEFB1 expression ratios.

QRT-PCR analysis of LCM normal tissue demonstrated that patients withrelative DEFB1 expression levels greater than 0.005 have a lower GleasonScore compared to those with expression levels lower than 0.005 (FIG.28A). Thus, there is an inverse relationship between DEFB1 expressionand Gleason score. Conversely, there was a positive correlation betweenPAX2 expression and Gleason score in malignant prostate tissue and PIN(FIG. 28B).

The PAX2 and DEFB1 expression levels in normal, PIN and canceroustissues from separate patients were calculated and compared (FIG. 29).Overall, PAX2 expression levels relative to GAPDH internal controlranged between 0 and 0.2 in normal (benign) tissue, 0.2 and 0.3 in PIN,and between 0.3 and 0.5 in cancerous (malignant) tissue (FIG. 30). ForDEFB1 there was an inverse relationship compared to PAX2. Here, DEFB1expression levels relative to GAPDH internal control ranged between 0.06and 0.005 in normal (benign) tissue, 0.005 and 0.003 in PIN, and between0.003 and 0.001 in cancerous (malignant) tissue. Therefore, disclosed isa predictive scale (designated as the “Donald predictive factor” or“DPF”) which utilizes the PAX2-DEFB1 expression ratio as aprognosticator of benign, precancerous (PIN) and malignant prostatetissue. Tissues with PAX2-DEFB1 ratios between 0 and 39 based on the DPFwill represent normal (pathologically benign). Tissue with a PAX2-DEFB1ratio between 40 and 99 will represent PIN (pre-cancerous) based on theDPF scale. Finally, tissue with a PAX2-DEFB1 ratio between 100 and 500will be malignant (low to high grade cancer).

There currently is a critical need for predictive biomarkers forprostate cancer development. It is known that the onset of prostatecancer occurs long before the disease is detectable by current screeningmethods such as the PSA test or the digital rectal exam. It is thoughtthat a reliable test which could monitor the progression and early onsetof prostate cancer would greatly reduce the mortality rate through moreeffective disease management. Disclosed herein is a predictive index toallow physicians to know well in advance the pathological state of theprostate. The DPF measures the decrease in the PAX2-DEFB1 expressionratio associated with prostate disease progression. This powerfulmeasure can not only predict the likelihood of a patient developingprostate cancer, but also may pinpoint the early onset of pre-malignantcancer. Ultimately, this tool can allow physicians to segregate whichpatients have more aggressive disease from those which do not.

The identification of cancer-specific markers has been utilized to helpidentify circulating tumor cells (CTCs). There is also emerging evidencewhich demonstrates that detection of tumor cells disseminated inperipheral blood can provide clinically important data for tumorstaging, prognostication, and identification of surrogate markers forearly assessment of the effectiveness of adjuvant therapy. Furthermore,by comparing gene expression profiling of all circulating cells, one canexamine the expression of the DEFB1 and PAX2 genes which play a role in“immunosurveillance” and “cancer survival”, respectively as aprognosticator for the early detection of prostate cancer.

Example 10 Functional Analysis of the Host Defense Peptide Human BetaDefensin-1: New Insight into its Potential Role in Cancer Materials andMethods

Tissue Samples and Laser Capture Microdissection:

Prostate tissues were obtained from patients who provided informedconsent prior to undergoing radical prostatectomy. Samples were acquiredthrough the Hollings Cancer Center tumor bank in accordance with anInstitutional Review Board-approved protocol. This included guidelinesfor the processing, sectioning, histological characterization, RNApurification and PCR amplification of samples. Prostate specimensreceived from the surgeons and pathologists were immediately frozen inOCT compound. Each OCT block was cut to produce serial sections whichwere stained and examined. Areas containing benign cells, prostaticintraepithelial neoplasia (PIN), and cancer were identified and used toguide our selection of regions from unstained slides using the ArcturusPixCell II System (Sunnyvale, Calif.). Caps containing captured materialwere exposed to 20 μl of lysate from the Arcturus Pico Pure RNAIsolation Kit and processed immediately. RNA quantity and quality wasevaluated using sets of primers that produce 5′ amplicons. The setsinclude those for the ribosomal protein L32 (the 3′ amplicon and the 5′amplicon are 298 bases apart), for the glucose phosphate isomerase (391bases apart), and for the glucose phosphate isomerase (842 bases apart).Ratios of 0.95 to 0.80 were routinely obtained for these primer setsusing samples from a variety of prepared tissues. Additional tumor andnormal samples were grossly dissected by pathologists, snap frozen inliquid nitrogen and evaluated for hBD-1 and cMYC expression.

Cloning of hBD-1 Gene:

hBD-1 cDNA was generated from RNA by reverse transcription-PCR usingprimers generated from the published hBD-1 sequence (accession no.U50930) (Ganz, 2004). The PCR primers were designed to contain ClaI andKpnI restriction sites. hBD-1 PCR products were restriction digestedwith ClaI and KpnI and ligated into a TA cloning vector. The TA/hBD1vector was then transfected into the XL-1 Blue strain of E. coli by heatshock and individual clones were selected and expanded. Plasmids wereisolated by Cell Culture DNA Midiprep (Qiagen, Valencia, Calif.) andsequence integrity verified by automated sequencing. The hBD-1 genefragment was then ligated into the pTRE2 digested with ClaI and KpnI,which served as an intermediate vector for orientation purposes. ThepTRE2/hBD-1 construct was digested with ApaI and KpnI to excise thehBD-1 insert. The insert was ligated into pIND vector of the EcdysoneInducible Expression System (Invitrogen, Carlsbad, Calif.) also doubledigested with ApaI and KpnI. The construct was transfected into E. coliand individual clones were selected and expanded. Plasmids were isolatedand sequence integrity of pIND/hBD-1 was again verified by automatedsequencing.

Transfection:

Cells (1×10⁶) were seeded onto 100-mm Petri dishes and grown overnight.Next, the cells were co-transfected using Lipofectamine 2000(Invitrogen) with 1 μg of pvgRXR plasmid, which expresses theheterodimeric ecdysone receptor, and 1 μg of the pIND/hBD-1 vectorconstruct or pIND/j-galactosidase (β-gal) control vector in Opti-MEMmedia (Life Technologies, Inc.). Transfection efficiency was determinedby inducing β-gal expression with Ponasterone A (PonA) and stainingcells with a β-galactosidase detection kit (Invitrogen). Assessment oftransfection efficiency by counting positive staining (blue) colonieswhich demonstrated that 60-85% of cells expressed β-galactosidase forthe cell lines.

Immunocytochemistry:

In order to verify hBD-1 protein expression, DU145 and hPrEC cells wereseeded onto 2-chamber culture slides (BD Falcon, USA) at 1.5-2×104 cellsper chamber. DU145 cells transfected with pvgRXR alone (control) or withthe hBD-1 plasmid were induced for 18 hours with media containing 10 μMPon A, while untransfected cells received fresh growth media. Followinginduction, cells were washed in 1×PBS and fixed for 1 hour at roomtemperature with 4% paraformaldehyde. Cells were then washed six timeswith 1×PBS and blocked in 1×PBS supplemented with 2% BSA, 0.8% normalgoat serum (Vector Laboratories, Inc., Burlingame, Calif.) and 0.4%Triton-X 100 for 1 hour at room temperature. Next, cells were incubatedovernight in primary rabbit anti-human BD-1 polyclonal antibody(PeproTech Inc., Rocky Hill, N.J.) diluted 1:1000 in blocking solution.Following this, cells were washed six times with blocking solution andincubated for 1 hour at room temperature in Alexa Fluor 488 goatanti-rabbit IgG (H+L) secondary antibody at a dilution of 1:1000 inblocking solution. After washing cells with blocking solution six times,coverslips were mounted with Gel Mount (Biomeda, Foster City, Calif.).Finally, cells were viewed under differential interference contrast(DIC) and under laser excitation at 488 nm. The fluorescent signal wasanalyzed by confocal microscopy (Zeiss LSM 5 Pascal) using a 63×DIC oillens with a Vario 2 RGB Laser Scanning Module. The digital images wereexported into Photoshop CS Software (Adobe Systems) for image processingand hard copy presentation.

RNA isolation and quantitative RT-PCR were performed as described inExample 1. The primer pairs for hBD-1 and c-MYC were generated from thepublished sequences (Table 6). Forty cycles of PCR were performed understandard conditions using an annealing temperature of 56.4° C. for hBD-1and c-MYC and 55° C. for PAX2. In addition, β-actin (Table 6) wasamplified as a housekeeping gene to normalize the initial content oftotal cDNA. Gene expression in benign prostate tissue samples wascalculated as the expression ratio compared to β-actin. Levels of hBD-1expression in malignant prostate tissue, hPREC prostate primary culture,and prostate cancer cell lines before and after induction werecalculated relative to the average level of hBD-1 expression in hPrECcells. As a negative control, QRT-PCR reactions without cDNA templatewere also performed. All reactions were run a minimum of three times.

MTT cell viability assay was performed as described in Example 1.

Analysis of membrane integrity was performed as described in Example 3.Cells transfected with empty plasmid or hBD-1 plasmid were induced for24 or 48 hours with media containing 10 μM Pon A, while control cellsreceived fresh growth media at each time point.

TABLE 6 Sequences of QRT-PCR primers Sense (5′-3′) Antisense (5′-3′) β-CCTGGCACCCAGCACAAT GCCGATCCACACGGAGTACT Actin (SEQ ID NO: 34)(SEQ ID NO: 36) hBD- TCAGCAGTGGAGGGCAATG CCTCTGTAACAGGTGCCTTGA 1(SEQ ID NO: 50) AT (SEQ ID NO: 51) cMYC ACAGCAAACCTCCTCACAGCTGGAGACGTGGCACCTCTTG C (SEQ ID NO: 53) (SEQ ID NO: 52) Nucleotidesequences of primers used to amplify hBD-1, cMyc, PAX2, and β-actin.

Flow cytometry and Caspase detection were performed as described inExample 1.

siRNA silencing of PAX2 was performed as described in Example 2. SiRNAmolecules were coated with CodeBreaker transfection reagent (Promega,Inc.) according to manufacturer's directions prior to treatment.

Statistical Analysis:

Statistical analysis was performed by using the Student's t-test forunpaired values. P values were determined by a two-sided calculation,and a P value of less than 0.05 was considered statisticallysignificant. Statistical differences are indicated by asterisks.

hBD-1 Expression in Prostate Tissue:

82% of prostate cancer frozen tissue sections analyzed exhibited littleor no expression of hBD-1 (Donald et al., 2003). To compare hBD-1expression levels, QRTPCR analysis was performed on normal prostatetissue obtained by gross dissection or LCM of normal prostate tissueadjacent to malignant regions which were randomly chosen. Here, hBD-1was detected in all of the gross dissected normal clinical samples witha range of expression that represents approximately a 6.6-folddifference in expression levels (FIG. 31A). LCM captured normal tissuesamples expressed hBD-1 at levels in a range that represents a 32-folddifference in expression (FIG. 31B). Matching sample numbers tocorresponding patient profiles revealed that in most cases, the hBD-1expression level was higher in patient samples with a Gleason score of 6than in patient samples with a Gleason score of 7. In addition, acomparison of hBD-1 expression levels in tissue obtained by grossdissection and LCM from the same patient, #1343, demonstrated an854-fold difference in expression between the two isolation techniques.Therefore, these results indicate that LCM provides a more sensitivetechnique to assess hBD-1 expression in prostate tissue.

hBD-1 Expression in Prostate Cell Lines:

To verify upregulation of hBD-1 in the prostate cancer cell lines aftertransfection with the hBD-1 expression system, QRTPCR was performed. Inaddition, no template negative controls were also performed, andamplification products were verified by gel electrophoresis. Here, hBD-1expression was significantly lower in the prostate cancer cell linescompared to hPrEC cells. Following a 24 hours induction period, relativeexpression levels of hBD-1 significantly increased in DU145, PC3 andLNCaP as compared to the cell lines prior to hBD-1 induction (FIG. 32A).

Next, protein expression of hBD-1 in was verified DU145 cellstransfected with the hBD-1 expression system after induction with Pon Aby immunocytochemistry. As a positive control, hBD-1 expressing hPrECprostate epithelial cells were also examined. Cells were stained withprimary antibody against hBD-1 and protein expression was monitoredbased on the green fluorescence of the secondary antibody (FIG. 32B).Analysis of cells under DIC verify the presence of hPrEC cells and DU145cells induced for hBD-1 expression at 18 hours. Excitation by theconfocal laser at 488 nm produced revealed green fluorescence indicatingthe presence of hBD-1 protein in hPrEC as a positive control. However,there was no detectable green fluorescence in control DU145 cells andempty plasmid induced DU145 cells demonstrating no hBD-1 expression.Confocal analysis of DU145 cells induced for hBD-1 expression revealedgreen fluorescence indicating the presence of hBD-1 protein followinginduction with Pon A.

Expression of hBD-1 Results in Decreased Cell Viability:

MTT assay was performed to assess the effect of hBD-1 expression onrelative cell viability in DU145, PC3, PC3/AR+ and LNCaP prostate cancercell lines. MTT analysis with empty vector exhibited no statisticalsignificant change in cell viability. Twenty-four hours following hBD-1induction, relative cell viability was 72% in DU145 and 56% in PC3cells, and after 48 hours cell viability was reduced to 49% in DU145 and37% in PC3 cells (FIG. 33A). Following 72 hours of hBD-1 induction,relative cell viability decreased further to 44% in DU145 and 29% PC3cells. Conversely, there was no significant effect on the viability ofLNCaP cells. In order to assess whether the resistance to hBD-1cytotoxicity observed in LNCaP was due to the presence of the androgenreceptor (AR), the hBD-1 cytotoxicity in PC3 cells was examined withectopic AR expression (PC3/AR+). Here, there was no difference betweenPC3/AR+ and PC3 cells. Therefore, the data indicates that that hBD-1 iscytotoxic specifically to late-stage prostate cancer cells.

In order to determine whether the effects of hBD-1 on PC3 and DU145 werecytostatic or cytotoxic, FACS analysis was performed to measure celldeath. Under normal growth conditions, more than 90% of PC3 and DU145cultures were viable and non-apoptotic (lower left quadrant) and did notstain with annexin V or PI (FIG. 4). After inducing hBD-1 expression inPC3 cells, the number of cells undergoing early apoptosis and lateapoptosis/necrosis (lower and upper right quadrants, respectively)totaled 10% at 12 hours, 20% at 24 hours, and 44% at 48 hours. For DU145cells, the number of cells undergoing early apoptosis and lateapoptosis/necrosis totaled 12% after 12 hours, 34% at 24 hours, and 59%after 48 hours of induction. No increase in apoptosis was observed incells containing empty plasmid following induction with Pon A. Annexin Vand propidium iodide uptake studies have demonstrated that hBD-1 hascytotoxic activity against DU145 and PC3 prostate cancer cells andresults indicate apoptosis as a mechanism of cell death.

hBD-1 causes alterations in membrane integrity and caspase activation:It was investigated whether the cell death observed in prostate cancercells after hBD-1 induction is caspase-mediated apoptosis. To betterunderstand the cellular mechanisms involved in hBD-1 expression,confocal laser microscopic analysis was performed (FIG. 5) on DU145 andLNCaP cells induced for hBD-1 expression. Pan-caspase activation wasmonitored based on the binding and cleavage of green fluorescingFAM-VAD-FMK to caspases in cells actively undergoing apoptosis. Analysisof cells under DIC showed the presence of viable control DU145 (FIG. 5A)and LNCaP (FIG. 5E) cells at Oh. Excitation by the confocal laser at 488nm produced no detectable green staining which indicates no caspaseactivity in DU145 (FIG. 5B) or LNCaP (FIG. 5F) control cells. Followinginduction for 24 hours, DU145 (FIG. 5C) and LNCaP (FIG. 5G) cells wereagain visible under DIC. Confocal analysis under fluorescence revealedgreen staining in DU145 (FIG. 5D) cells indicating pan-caspase activityafter the induction of hBD-1 expression. However, there was no greenstaining in LNCaP (FIG. 5H) cells induced for hBD-1 expression.Therefore, cell death observed following induction of hBD-1 iscaspase-mediated apoptosis.

The proposed mechanism of antimicrobial activity of defensin peptides isthe disruption of the microbial membrane due to pore formation (Papo andShai, 2005). In order to determine if hBD-1 expression altered membraneintegrity EtBr uptake was examined by confocal analysis. Intact cellswere stained green due to AO which is membrane permeable, while onlycells with compromised plasma membranes stained red due to incorporationof membrane impermeable EtBr. Control DU145 and PC3 cells stainedpositively with AO and emitted green color, but did not stain with EtBr.However, hBD-1 induction in both DU145 and PC3 resulted in theaccumulation of EtBr in the cytoplasm at 24 as indicated by the redstaining. By 48 hours, DU145 and PC3 possessed condensed nuclei andappeared yellow due to the colocalization of green and red staining fromAO and EtBr, respectively. Conversely, there were no observablealterations to membrane integrity in LNCaP cells after 48 hours ofinduction as indicated by positive green fluorescence with AO, but lackof red EtBr fluorescence. This finding indicates that alterations tomembrane integrity and permeablization in response to hBD-1 expressiondiffer between early- and late-stage prostate cancer cells.

Comparison of hBD-1 and cMYC Expression Levels:

QRT-PCR analysis was performed on LCM prostate tissue sections fromthree patients (FIG. 34). In patient #1457, hBD-1 expression exhibited a2.7-fold decrease from normal to PIN, a 3.5-fold decrease from PIN totumor and a 9.3-fold decrease from normal to tumor (FIG. 34A). Likewise,cMYC expression followed a similar expression pattern in patient #1457where expression decreased by 1.7-fold from normal to PIN, 1.7-fold fromPIN to tumor and 2.8-fold from normal to tumor (FIG. 34B). In addition,there was a statistically significant decrease in cMYC expression in theother two patients. Patient #1569 had a 2.3-fold decrease from normal toPIN, while in patient #1586 there was a 1.8-fold decrease from normal toPIN, a 4.3-fold decrease from PIN to tumor and a 7.9-fold decrease fromnormal to tumor.

Induction of hBD-1 Expression Following PAX2 Inhibition:

To further examine the role of PAX2 in regulating hBD-1 expression,siRNA was utilized to knockdown PAX2 expression and QRT-PCR performed tomonitor hBD-1 expression. Treatment of hPrEC cells with PAX2 siRNAexhibited no effect on hBD-1 expression (FIG. 35). However, PAX2knockdown resulted in a 42-fold increase in LNCaP, a 37-fold increase inPC3 and a 1026-fold increase in DU145 expression of hBD-1 compared tountreated cells. As a negative control, cells were treated withnon-specific siRNA which had no significant effect on hBD-1 expression.

Example 11 Inhibition of PAX2 Expression Results in Alternate Cell DeathPathways in Prostate Cancer Cells Differing in P53 Status Materials andMethods

Cell Lines:

The cancer cell lines PC3, DU145 and LNCaP, which all differ in p53mutational status (Table 7) were cultured as described in Example 1.

siRNA silencing of PAX2 and Western analysis were performed as describedin Example 2. Blots were then probed with rabbit anti-PAX2 primaryantibody (Zymed, San Francisco, Calif.) at a 1:1000 dilution. Afterwashing, the membranes were incubated with anti-rabbit antibodyconjugated to horseradish peroxidase (HRP) (dilution 1:5000; Sigma), andsignal detection was visualized using chemiluminescence reagents(Pierce) on an Alpha Innotech Fluorchem 8900. As a control, blots werestripped and reprobed with mouse anti-β-actin primary antibody (1:5000;Sigma-Aldrich) and HRP-conjugated anti-mouse secondary antibody (1:5000;Sigma-Aldrich), and signal detection was again visualized.

TABLE 7 p53 gene mutation in prostate cancer cell lines Amino CellNucleotide acid Gene line change change status Reference DU145 CCT-CTTPro-Leu Gain/loss- Tepper et al. 2005; of-function Bodhoven et al. 2003GTT-TTT Val-Phe PC3 Deleted a C, Frame- No activity Isaacs et al. 1991GCC-GC shift LNCaP No deletion, — Normal Carroll et al. 1993 wild-typefunction

Phase contrast microscopy and MTT cytotoxicity assay were performed asdescribed in Example 2.

Pan-caspase detection and Quantitative real-time RT-PCR were performedas described in Example 1. The primer pairs for BAX, BID, BCL-2, AKT andBAD were generated from the published sequences (Table 8). Reactionswere performed in MicroAmp Optical 96-well Reaction Plate (PEBiosystems). Forty cycles of PCR were performed under standardconditions using an annealing temperature of 60° C. Quantification wasdetermined by the cycle number where exponential amplification began(threshold value) and averaged from the values obtained from thetriplicate repeats. There was an inverse relationship between messagelevel and threshold value. In addition, GAPDH was used as a housekeepinggene to normalize the initial content of total cDNA. Relative expressionwas calculated as the ratio between each genes and GAPDH. All reactionswere carried out in triplicate.

TABLE 8 Quantitative RT-PCR primers Sense (5′-3′) Antisense (5′-3′)GAPDH CCACCCATGGCAAATTCCAT TCTAGACGGCAGGTCAGGTC GGCA (SEQ ID NO: 42)AACC (SEQ ID NO: 46) BAD CTCAGGCCTATGCAAAAAGA GCCCTCCCTCCAAAGGAGACGGA (SEQ ID NO: 43) (SEQ ID NO: 47) BID AACCTACGCACCTACGTGAGCGTTCAGTCCATCCCATTTC GAG (SEQ ID NO: 44) TG (SEQ ID NO: 48) BAXGACACCTGAGCTGACCTTGG GAGGAAGTCCAGTGTCCAGC (SEQ ID NO: 45)(SEQ ID NO: 49) BCL-2 TATGATACCCGGGAGATCGT GTGCAGATGCCGGTTCAGGTAGATC (SEQ ID NO: 54) CTC (SEQ ID NO: 55) AKT TCAGCCCTGGACTACCTGCAGAGGTCCCGGTACACCACGT (SEQ ID NO: 56) (SEQ ID NO: 57)

Membrane permeability assay was performed as described in Example 3. PC3and LNCaP cells were transfected with PAX2 siRNA, non-specific siRNA ormedia only.

Analysis of PAX2 Protein Expression in Prostate Cells:

PAX2 protein expression was examined by Western analysis in HPrECprostate primary culture and in LNCaP, DU145 and PC3 prostate cancercell lines. Here, PAX2 protein was detected in all of the prostatecancer cell lines (FIG. 36A). However, no PAX2 protein was detectable inHPrEC. Blots were stripped and re-probed for β-actin as internal controlto ensure equal loading. PAX2 protein expression was also monitoredafter selective targeting and inhibition by PAX2 specific siRNA inDU145, PC3 and LNCaP prostate cancer cell lines. Cells were given asingle round of transfection with the pool of PAX2 siRNA over a 6-daytreatment period. PAX2 protein was expressed in control cells treatedwith media only. Specific targeting of PAX2 mRNA was confirmed byobserving knockdown of PAX2 protein in all three cell lines (FIG. 36B).

Effect of PAX2 Knockdown on Prostate Cancer Cell Growth:

The effect of PAX2 siRNA on cell number and cell viability was analyzedusing light microscopy and MTT analysis. To examine the effect of PAX2siRNA on cell number, PC3, DU145 and LNCaP cell lines were transfectedwith media only, non-specific siRNA or PAX2 siRNA over a period of 6days. Each of the cell lines reached a confluency of 80-90% in 60 mmculture dishes containing media only. Treatment of HPrEC, DU145, PC3 andLNCaP cells with non-specific siRNA appeared to have little to no effecton cell growth compared to cell treated with media only (FIGS. 38A, 38Cand 38E, respectively). Treatment of the PAX2-null cell line HPrEC withPAX2 siRNA appeared to have no significant effect on cell growth (FIG.37B). However, treatment of the prostate cancer cell lines DU145, PC3and LNCaP with PAX2 siRNA resulted in a significant decrease in cellnumber (FIGS. 38D, 38F and 38H, respectively).

Effect of PAX2 Knockdown on Prostate Cancer Cell Viability:

Cell viability was measured after 2-, 4-, and 6-day exposure times.Percent viability was calculated as the ratio of the 570-630 nmabsorbance of cell treated with PAX2 siRNA divided by untreated controlcells. As negative controls, cell viability was measured after eachtreatment period with negative control non-specific siRNA ortransfection with reagent alone. Relative cell viability was calculatedby dividing percent viability following PAX2 siRNA treatment by percentviability following treatment with non-specific shRNA (FIG. 38). After 2days of treatment, relative viability was 116% in DU145, 81% in PC3 and98% in LNCaP. After 4 days of treatment, relative cell viabilitydecreased to 69% in DU145, 79% in PC3, and 80% in LNCaP. Finally, by 6days relative viability was 63% in DU145, 43% in PC3 and 44% in LNCaP.In addition, cell viability was also measured following treatment withtransfection reagent alone. Here, each cell line exhibited nosignificant decrease in cell viability.

Detection of Pan-Caspase Activity:

Caspase activity was detected by confocal laser microscopic analysis.LNCaP, DU145 and PC3 cells were treated with PAX2 siRNA and activity wasmonitored based on the binding of FAM-labeled peptide to caspases incells actively undergoing apoptosis which will be fluoresce green.Analysis of cells with media only shows the presence of viable LNCaP,DU145 and PC3 cells, respectively. Excitation by the confocal laser at488 nm produced no detectable green staining which indicates no caspaseactivity in the untreated cells (FIGS. 39A, 39C and 39E, respectively).Following 4 days of treatment with PAX2 siRNA, LNCaP, DU145 and PC3cells under fluorescence presented green staining indicating caspaseactivity (FIGS. 39B, 39D and 39F, respectively).

Effect of PAX2 Inhibition on Apoptotic Factors:

LNCaP, DU145 and PC3 cells were treated with siRNA against PAX2 for 4days and expression of both pro- and anti-apoptotic factors weremeasured by QRTPCR. Following PAX2 knockdown, analysis of BAD revealed a2-fold in LNCaP, 1.58-fold in DU145 and 1.375 in PC3 (FIG. 40A).Expression levels of BID increased by 1.38-fold in LNCaP and a 1.78-foldincrease in DU145, but there was no statistically significant differencein BID observed in PC3 after suppressing PAX2 expression (FIG. 40B).Analysis of the anti-apoptotic factor AKT revealed a 1.25-fold decreasein expression in LNCaP and a 1.28-fold decrease in DU145 followingtreatment, but no change was observed in PC3 (FIG. 40C).

Analysis of Membrane Integrity and Necrosis:

Membrane integrity was monitored by confocal analysis in LNCaP, DU145and PC3 cells. Here, intact cells stained green due to AO which ismembrane permeable, while cells with compromised plasma membranes wouldstained red due to incorporation of membrane impermeable EtBr into thecytoplasm, and yellow due to co-localization of AO and EtBr in thenuclei. Untreated LNCaP, DU145 and PC3 cells stained positively with AOand emitted green color, but did not stain with EtBr. Following PAX2knockdown, there were no observable alterations to membrane integrity inLNCaP cells as indicated by positive green fluorescence with AO andabsence of red EtBr fluorescence. These finding further indicate thatLNCaP cells can be undergoing apoptotic, but not necrotic cell deathfollowing PAX2 knockdown. Conversely, PAX2 knockdown in DU145 and PC3resulted in the accumulation of EtBr in the cytoplasm as indicated bythe red staining. In addition, both DU145 and PC3 possessed condensednuclei which appeared yellow due to the co-localization of green and redstaining from AO and EtBr, respectively. These results indicate thatDU145 and PC3 are undergoing an alternate cell death pathway involvingnecrotic cell death compared to LNCaP.

Example 12 PAX2 and DEFB-1 Expression in Breast Cancer Cell Lines AndMammary Tissues with Ductal or Lobular Intraepithelial Neoplasia

PAX2 and DEFB-1 expression will be determined in breast biopsy samplesof ductal or lobular intraepithelial neoplasia, and in the followingbreast cancer cell lines: BT-20: Isolated from a primary invasive ductalcarcinoma; cell express E-cadherin, ER, EGFR and uPA.

BT-474: Isolated from a primary invasive ductal carcinoma; cell expressE-cadherin, ER, PR, and have amplified HER2/neu.

Hs578T: Isolated from a primary invasive ductal carcinoma; a cell linewas also established from normal adjacent tissue, termed Hs578Bst.

MCF-7: Established from a pleural effusion. The cells express ER and arethe most common example of estrogen-responsive breast cancer cells.

MDA-MB-231: Established from a pleural effusion. The cells areER-negative, E-cadherin negative and highly invasive in in vitro assays.

MDA-MB-361: Established from a brain metastasis. The cells express ER,PR, EGFR and HER2/neu.

MDA-MB-435: Established from a pleural effusion. The cells areER-negative, E-cadherin negative, and are highly invasive and metastaticin immunodeficient mice.

MDA-MB-468: Established from a pleural effusion. The cells haveamplified EGFR and are ER-negative.

SK-BR-3: Established from a pleural effusion. The cells have amplifiedHER/2neu, express EGFR and are ER-negative.

T-47D: established from a pleural effusion. The cells retain expressionof E-cadherin, ER and PR.

ZR-75-1: Established from ascites fluid. The cells express ER,E-cadherin, HER2/neu and VEGF.

The PAX2-to-DEFB-1 expression ratio will be determined using the methodsdescribed in Example 9.

Example 13 Expression of DEFB1 in Breast Cancer Cells

DEFB1 will be expressed in breast cancer cells using methods describedin Example 1. The cell viability and caspase activity will be determinedas described in Example 1.

Example 14 Inhibition Of PAX2 Expression in Breast Cancer Cells

PAX2 expression in breast cancer cells will be inhibited using the siRNAdescribed in Example 2. The expression levels of pro-apoptotic genessuch as BAX, BID and BAD, the cell viability and caspase activity willbe determined as described in Example 2.

Example 15 Effect of DEFB1 expression on tumor growth in vivo

The anti-tumoral ability of DEFB1 will be evaluated by injecting breastcancer cells that overexpress DEFB1 into nude mice. Breast cancer cellswill be transfected with an expression vector carrying the DEFB1 gene.Cells expressing the exogenous DEFB1 gene will be selected and cloned.Only single-cell suspensions with a viability of >90% are used. Eachanimal receives approximately 500,000 cells administered subcutaneouslyinto the right flank of female nude mice. There are two groups, acontrol group injected with vector only clones and a group injected withthe DEFB1 over-expressing clones. 35 mice are in each group asdetermined by a statistician. Animals are weighed twice weekly, tumorgrowth monitored by calipers and tumor volumes determined using thefollowing formula: volume=0.5×(width)2×length. All animals aresacrificed by CO2 overdose when tumor size reaches 2 mm3 or 6 monthsfollowing implantation; tumors are excised, weighed and stored inneutral buffered formalin for pathological examination. Differences intumor growth between the groups are descriptively characterized throughsummary statistics and graphical displays. Statistical significance isevaluated with either the t-test or non-parametric equivalent.

Example 16 Effect of PAX2 siRNA on tumor growth in vivo

Hairpin PAX2 siRNA template oligonucleotides utilized in the in vitrostudies are utilized to examine the effect of the up-regulation of DEFB1expression in vivo. The sense and antisense strand (see Table 3) areannealed and cloned into pSilencer 2.1 U6 hygro siRNA expression vector(Ambion) under the control of the human U6 RNA pol III promoter. Thecloned plasmid is sequenced, verified and transfected into breast cancercell lines. Scrambled shRNA is cloned and used as a negative control inthis study. Hygromycin resistant colonies are selected, cells areintroduced into the mice subcutaneously and tumor growth is monitored asdescribed above.

Example 17 Effect of Small Molecule Inhibitors of PAX2 Binding on BreastCancer Cells

The alternative inhibitory oligonucleotides described in Example 6 willbe transfected into the breast cancer cells with lipofectamine reagentor Codebreaker transfection reagent (Promega, Inc). In order to confirmDNA-protein interactions, double stranded oligonucleotides will belabeled with [³²P] dCTP and electrophoretic mobility shift assays areperformed DEFB1 expression will be monitored by QRT-PCR and Westernanalysis following treatment with oligonucleotides. Finally, cell deathwill be detected by MTT assay and flow cytometry as previouslydescribed.

The above description is for the purpose of teaching the person ofordinary skill in the art how to practice the present invention, and itis not intended to detail all those obvious modifications and variationsof it which will become apparent to the skilled worker upon reading thedescription. It is intended, however, that all such obviousmodifications and variations be included within the scope of the presentinvention, which is defined by the following claims. The claims areintended to cover the claimed components and steps in any sequence whichis effective to meet the objectives there intended, unless the contextspecifically indicates the contrary.

1. A method for monitoring breast conditions in a subject, comprising: determining an expression level of a Paired Box 2 (PAX2) gene relative to an expression level of a control gene in cells obtained from a breast of the subject; determining an expression level of a beta defensin-1 (DEFB1) gene relative to the expression level of the control gene in the cells; and determining a PAX2-to-DEFB1 expression ratio based on the expression levels of PAX2 and DEFB1 in the cells, wherein the PAX2-to-DEFB1 expression ratio is correlated with breast conditions and wherein a PAX 2-to-DEFB1 expression ratio of 100:1 or higher is indicative of the presence of breast cancer in the subject, and a PAX2-to-DEFB1 expression ratio of less than 100:1 is indicative of the presence of non-cancerous or pre-cancerous breast condition in the subject.
 2. (canceled)
 3. The method of claim 1, wherein the pre-cancerous breast condition is mammary intraepithelial neoplasia.
 4. (canceled)
 5. The method of claim 1, wherein the expression levels of PAX2, DEFB1 and the control genes are determined by quantitative real-time RT-PCR.
 6. The method of claim 1, wherein the expression levels of PAX2, DEFB1 and the control genes are determined by expression microarray.
 7. The method of claim 1, wherein the control gene is the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene.
 8. The method of claim 1, further comprising determining an oestrogen receptor/progesterone receptor/human epidermal growth factor receptor 2 (ER/PR/HER2) status in the cells obtained from the breast tissue. 9-24. (canceled)
 25. A method for monitoring breast conditions in a subject, comprising: extracting RNA from cells obtained from a breast of a subject to produce an extracted RNA sample; determining an expression level of a Paired Box 2 (PAX2) gene relative to an expression level of a control gene in the extracted RNA sample; determining an expression level of a beta defensin-1 (DEFB1) gene relative to the expression level of the control gene in the extracted RNA sample; and determining a PAX2-to-DEFB1 expression ratio based on the expression levels of PAX2 and DEFB1 in the extracted RNA sample; correlating the PAX2-to-DEFB1 expression ratio with breast conditions, wherein a PAX 2-to-DEFB1 expression ratio of 100:1 or higher is indicative of the presence of breast cancer in the subject.
 26. The method of claim 25, wherein the pre-cancerous breast condition is mammary intraepithelial neoplasia.
 27. The method of claim 25, wherein the expression levels of PAX2, DEFB1 and the control genes are determined by quantitative real-time RT-PCR.
 28. The method of claim 25, wherein the expression levels of PAX2, DEFB1 and the control genes are determined by expression microarray.
 29. The method of claim 25, wherein the control gene is the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene.
 30. The method of claim 25, further comprising determining an oestrogen receptor/progesterone receptor/human epidermal growth factor receptor 2 (ER/PR/HER2) status in the cells obtained from the breast of the subject.
 31. A method for detecting breast cancer in a subject, comprising: extracting proteins from cells obtained from a breast of a subject to produce an extracted protein sample; determining an expression level of a Paired Box 2 (PAX2) gene relative to an expression level of a control gene in the extracted protein sample; determining an expression level of a beta defensin-1 (DEFB1) gene relative to the expression level of the control gene in the protein sample; and determining a PAX2-to-DEFB1 expression ratio based on the expression levels of PAX2 and DEFB1 in the extracted protein sample; correlating the PAX2-to-DEFB1 expression ratio with breast conditions, wherein a PAX 2-to-DEFB1 expression ratio of 100:1 or higher is indicative of the presence of breast cancer in the subject.
 32. The method of claim 31, wherein the pre-cancerous breast condition is mammary intraepithelial neoplasia.
 33. The method of claim 31, wherein the expression levels of PAX2, DEFB1 and the control gene are determined by immunoassays.
 34. The method of claim 31, wherein the expression levels of PAX2, DEFB1 and the control gene are determined by radioimmune precipitation assay (RIPA).
 35. The method of claim 31, wherein the expression levels of PAX2, DEFB1 and the control gene are determined by an immunoassays wherein an antibody specific for PAX2, DEFB1 or the control gene is bound to a solid support.
 36. The method of claim 31, wherein the control gene is the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene.
 37. The method of claim 31, further comprising determining an oestrogen receptor/progesterone receptor/human epidermal growth factor receptor 2 (ER/PR/HER2) status in the cells obtained from the breast of the subject. 